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.     

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.     

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.     

Inhibition of morphine analgesia by LPS: role of opioid and NMDA receptors and spinal glia

Intraperitoneal (i.p.) injection of toxins, such as the bacterial endotoxin lipopolysaccharide (LPS), is associated with a well-characterized increase in sensitivity to painful stimuli (hyperalgesia) [Watkins LR, Maier SF, Goehler LE. Immune activation: the role of pro-inflammatory cytokines in inflammation, illness responses and pathological pain states. Pain 1995;63:289-302. [53]] and a longer-lasting reduction in opioid analgesia (anti-analgesia) when pain sensitivity returns to basal levels [Johnston IN, Westbrook RF. Acute and conditioned sickness reduces morphine analgesia. Behav Brain Res 2003;142:89-97]. Here we show that this inhibition of morphine analgesia 24 h after a single i.p. injection of LPS involves mechanisms that contribute to illness-induced hyperalgesia and the development of analgesic tolerance to morphine. Specifically, morphine analgesia was restored if LPS was preceded by systemic administration of a non-competitive NMDA receptor antagonist (MK-801), spinal infusion of a glial metabolic inhibitor (fluorocitrate), or intracerebroventricular microinjection of an opioid receptor antagonist (naloxone). Morphine analgesia was also restored if MK-801 was administered after LPS. These results demonstrate that LPS recruits similar, if not the same mechanisms that reduce morphine tolerance following opiate administration: namely, stimulation of opioid and NMDA receptors and recruitment of spinal glia.     

Glial activation in the rostroventromedial medulla promotes descending facilitation to mediate inflammatory hypersensitivity

Substantial evidence shows that activation of glial cells in the spinal cord may promote central sensitization and pain. Descending facilitation from the rostroventromedial medulla (RVM) is a critical component in the maintenance of chronic pain states, although the precise mechanisms through which facilitation maintains pain are unclear. Here, we investigated the possibility that glial activation in the RVM could promote descending facilitation from the RVM in states of inflammatory pain. Peripheral inflammation was induced with carrageenan injected into the hindpaws of male Sprague–Dawley rats, and behavioral responses to noxious thermal and light tactile stimuli were determined. Microinjection of the glial inhibitors minocycline or fluorocitrate, or of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580, produced a significant and time-related attenuation of behavioral hypersensitivity resulting from hindpaw inflammation. Carrageenan-induced inflammation increased immunolabeling for microglia and astrocytes in the RVM, as well as for phosphorylated p38 MAPK. Phosphorylated p38 MAPK was found in microglia and neurons of the RVM. Inflammation-induced microglial and astrocytic activation in the RVM were attenuated by RVM microinjection of the glial inhibitors. The data show that inflammatory pain is associated with glial activation in the RVM that probably participates in driving descending pain facilitation. These findings reveal a novel site of glial modulation of inflammatory pain.     

Ligustilide attenuates inflammatory pain via inhibition of NFκB‐mediated chemokines production in spinal astrocytes

Ligustilide (LIG) is a major component of Radix Angelica Sinensis, and reportedly has neuroprotective and anti‐inflammatory effects. Recent studies have demonstrated that spinal astrocyte‐mediated neuroinflammation plays an important role in the pathogenesis of chronic pain. Here we investigated the anti‐nociceptive effect of systemic treatment with LIG on chronic inflammatory pain and explored possible mechanisms. Unilateral hindpaw injection of complete Freund's adjuvant (CFA) induced persistent pain hypersensitivity. Repeated daily intravenous treatment with LIG, either before or after CFA injection, attenuated CFA‐induced thermal hyperalgesia and mechanical allodynia. The same treatment also inhibited CFA‐induced keratinocyte‐derived chemokine (KC) and monocyte chemoattractant protein‐1 (MCP‐1) mRNA and protein increases in astrocytes of the spinal cord. In vitro study showed LIG dose‐dependently reduced lipopolysaccharide (LPS)‐induced upregulation of KC and MCP‐1 mRNA in astrocyte cultures. Interestingly, LIG treatment did not affect CFA‐ or LPS‐induced glial fibrillary acidic protein upregulation, but did inhibit CFA‐induced phosphorylated nuclear factor‐κB (p‐NFκB) upregulation in spinal astrocytes. Furthermore, intrathecal injection of NFκB inhibitor attenuated CFA‐induced pain hypersensitivity and upregulation of KC and MCP‐1 in the spinal cord. Finally, single intravenous injection of LIG attenuated intrathecal injection of LPS‐induced mechanical allodynia. The same treatment also decreased LPS‐induced NFκB activation and KC and MCP‐1 upregulation in the spinal cord. These data indicate that LIG attenuates chronic inflammatory pain potentially via inhibiting NFκB‐mediated chemokines production in spinal astrocytes. These results provide direct evidence of the anti‐nociceptive and anti‐inflammatory effects of LIG, suggesting a new application of LIG for the treatment of chronic inflammatory pain.     

Chemical stimulation of the ST36 acupoint reduces both formalin-induced nociceptive behaviors and spinal astrocyte activation via spinal alpha-2 adrenoceptors

Spinal astrocytes have emerged as important mechanistic contributors to pathological and chronic pain. Recently, we have demonstrated that injection of diluted bee venom (DBV) into the Zusanli (ST36) acupoint produces a potent anti-nociceptive effect via the activation of spinal alpha-2 adrenoceptors. However, it is unclear if this anti-nociceptive effect is associated with alterations in spinal astrocytes. Thus, the present study was designed to determine: (1) whether DBV's anti-nociceptive effect in the formalin test involves suppression of spinal astrocyte activation; (2) whether DBV-induced astrocyte inhibition is mediated by spinal alpha-2 adrenoceptors; and (3) whether this glial modulation is potentiated by intrathecal administration of the glial metabolic inhibitor, fluorocitrate (FC) in combination with DBV injection. DBV was injected directly into the ST36 acupoint, and spinal expression of the astrocytic marker, glial fibrillary acidic protein (GFAP), was assessed together with effects on formalin-induced nociception. DBV treatment reduced pain responses in the late phase of the formalin test and significantly blocked the formalin-evoked increase in spinal GFAP expression. These effects of DBV were prevented by intrathecal pretreatment with selective alpha-2A and alpha-2C adrenoceptor antagonists. Moreover, low dose intrathecal injection of FC in conjunction with low dose DBV injection into the ST36 acupoint synergistically suppressed pain responses and GFAP expression. These results demonstrate that DBV stimulation of the ST36 acupoint inhibits the formalin-induced activation of spinal astrocytes and nociceptive behaviors in this inflammatory pain model and this inhibition is associated with the activation of spinal alpha-2 adrenoceptors.     

Upregulation of Chemokine CXCL12 in the Dorsal Root Ganglia and Spinal Cord Contributes to the Development and Maintenance of Neuropathic Pain Following Spared Nerve Injury in Rats

Emerging evidence indicates that CXCL12/CXCR4 signaling is involved in chronic pain. However, few studies have systemically assessed its role in direct nerve injury-induced neuropathic pain and the underlying mechanism. Here, we determined that spared nerve injury(SNI)increased the expression of CXCL12 and its cognate receptor CXCR4 in lumbar 5 dorsal root ganglia(DRG)neurons and satellite glial cells. SNI also induced longlasting upregulation of CXCL12 and CXCR4 in the ipsilateral L4–5 spinal cord dorsal horn, characterized by CXCL12 expression in neurons and microglia, and CXCR4 expression in neurons and astrocytes. Moreover, SNIinduced a sustained increase in TNF-a expression in the DRG and spinal cord. Intraperitoneal injection(i.p.) of the TNF-a synthesis inhibitor thalidomide reduced the SNI-induced mechanical hypersensitivity and inhibited the expression of CXCL12 in the DRG and spinal cord.Intrathecal injection(i.t.) of the CXCR4 antagonist AMD3100, both 30 min before and 7 days after SNI,reduced the behavioral signs of allodynia. Rats given an i.t.or i.p. bolus of AMD3100 on day 8 of SNI exhibited attenuated abnormal pain behaviors. The neuropathic pain established following SNI was also impaired by i.t. administration of a CXCL12-neutralizing antibody. Moreover,repetitive i.t. AMD3100 administration prevented the activation of ERK in the spinal cord. The mechanical hypersensitivity induced in na?¨ve rats by i.t. CXCL12 was alleviated by pretreatment with the MEK inhibitor PD98059. Collectively, our results revealed that TNF-a might mediate the upregulation of CXCL12 in the DRG and spinal cord following SNI, and that CXCL12/CXCR4 signaling via ERK activation contributes to the development and maintenance of neuropathic pain.     

Electroacupuncture attenuates mechanical and warm allodynia through suppression of spinal glial activation in a rat model of neuropathic pain

Neuropathic pain remains one of the most difficult clinical pain syndromes to treat. It is traditionally viewed as being mediated solely by neurons; however, glial cells have recently been implicated as powerful modulators of pain. It is known that the analgesic effects of electroacupuncture (EA) are mediated by descending pain inhibitory systems, which mainly involve spinal opioid, adrenergic, dopaminergic, serotonergic, and cholinergic receptors. However, studies investigating the suppressive effects of EA on spinal glial activation are rare. In the present study, we assessed the cumulative analgesic effects of EA on mechanical and warm allodynia in a rat model of neuropathic pain. We investigated the clinical efficacy of EA as long-term therapy and examined its effects on spinal glia, matrix metalloproteinase (MMP)-9/MMP-2, proinflammatory cytokines and serum immunoglobulin G (IgG) concentration. Rats were randomly divided into four groups as follows: the operation group (OP), operation with EA-non acupoint (EA-NA), operation with EA-ST36 acupoint (EA-ST36), and sham operation (shamOP). Following neuropathic or sham surgery, repeated EA was performed every other day after the behavioral test. On day 53 after the behavioral test, rats were perfused for immunohistochemistry and Western blot analysis to observe quantitative changes in spinal glial markers such as OX-42, astrocytic glial fibrillary acidic protein (GFAP), MMP-9/MMP-2, and proinflammatory cytokines. Allodynia and OX-42/GFAP/MMP-9/MMP-2/tumor necrosis factor (TNF)-α/interleukin (IL)-1β activity in the EA-ST36 group was significantly reduced, compared to the OP and EA-NA groups, and IgG in EA-ST36 rats significantly increased. Our results suggest that the analgesic effect of EA may be partly mediated via inhibition of inflammation and glial activation and repeated EA stimulation may be useful for treating chronic pain clinically.     

Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring 5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.     

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.     

The effect of intrathecal administration of glial activation inhibitors on dorsal horn BDNF overexpression and hind paw mechanical allodynia in spinal nerve ligated rats

Recent studies have suggested that activated glia in the spinal cord may play a vital role at different times during spinal nerve ligation (SNL)-induced neuropathic pain; therefore, glial activation inhibitors have been used as effective painkillers. Brain-derived neurotrophic factor (BDNF) is also known to be a powerful pain modulator, but it remains unclear how it contributes to the glial activation inhibitor-based treatment. This study revealed the following results: (1) intrathecal administration of minocycline (a microglial activation inhibitor) could prevent mechanical allodynia during the initiation of SNL-induced neuropathic pain, and its action was associated with the elimination of BDNF overexpression in the dorsal horn; (2) the spinal injection of fluorocitrate (an astrocytic activation inhibitor) but not minocycline could reverse mechanical allodynia during the maintenance phase of SNL-induced pain, and its action was also related to a decrease in BDNF overexpression in the dorsal horn; and (3) treatment with TrkB/Fc (a BDNF-sequestering protein) had a similar effect during both the early development and maintenance periods. These results led to the following conclusions: (1) elevated BDNF expression in the dorsal horn was required to develop and maintain neuropathic pain; (2) minocycline could only prevent mechanical allodynia in the early stages, possibly by inhibiting BDNF release from microglia; and (3) fluorocitrate could reverse existing mechanical allodynia, and its action was associated with the inhibition of BDNF upregulation induced by astrocytic activation.     

Antiallodynic effects of propentofylline Elicited by interrupting spinal glial function in a rat model of bone cancer pain

The activation of microglia and astrocytes in the spinal cord is involved in the progress of cancer pain. Propentofylline (PPF), a glial modulating agent, alleviates pain hypersensitivity in neuropathic pain models. The present study investigated the potential roles of PPF in a preclinical rat model of bone caner pain established by inoculating Walker 256 cells into the left tibia. At day 9 postinoculation, single administration of PPF (10 μg/10 μl, i.t.) significantly but transiently suppressed mechanical allodynia induced by bone cancer. Repeated application of PPF (10 μg/10 μl, i.t., once daily from days 9 to 12) persistently relieved mechanical allodynia on the side ipsilateral to surgery. Immunohistochemistry and ELISA showed that microglia and astrocytes in the spinal cord were activated, and the production of glia-derived proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) markedly increased at day 12 postinoculation in the cancer group. Intrathecal injection of PPF (10 μg/10 μl) significantly inhibited the activation of spinal glial cells and the expression of proinflammatory cytokines. These results suggest that the glial modulating agent PPF has antiallodynic effects on bone cancer pain and has potential utility for clinical treatment of cancer pain.     

Nonsteroidal anti-inflammatory drugs increase expression of inducible COX-2 isoform of cyclooxygenase in spinal cord of rats with adjuvant induced inflammation

Several lines of evidence have accumulated that release of excitatory amino acids, nitric oxide and prostaglandin E2 (PGE2) play a critical role in the development of peripheral tactile and thermal hypersensitivity in chronic inflammatory pain models. Synthesis of PGE2 is controlled by cyclooxygenase (COX), either the COX-1 or COX-2 isoform. COX-2 plays a central role in the inflammatory reactions. The relationship between central sensitization of a complete Freund's adjuvant (CFA) induced inflammation and expressions of COX-2 were assessed in a rat model of CFA injection induced inflammation. Moreover, the time course of analgesia and spinal COX-2 expression following intrathecal (IT) injection with a nonspecific COX inhibitor (ketorolac) and COX-2 inhibitor (celecoxib) were determined using Western blot and immunohistochemistry. COX-2 protein was slightly increased in the lumbosacral spinal cord at 24 h following subcutaneous injection of CFA in the plantar surface of the left hindpaw (p > 0.05). COX-1 was not detected in normal and CFA injection rats. Surprisingly, IT ketorolac or celecoxib significantly increased spinal COX-2 levels at 1 h post-IT injection (p < 0.05) both in inflamed and non-inflamed rats. Then, spinal COX-2 levels declined at 3 and 6 h post-IT injection. These results provide strong in vivo evidence that COX-2 activity but not level may play a central role in the Freund's adjuvant-induced inflammation. However, spinal COX-2 level was upregulated following IT ketorolac and celecoxib injection. These data implies that suppression of PGE2 activity may induce the expression of spinal COX-2 in Freund's adjuvant-induced pain model. Our study concludes that IT administration of COX-2 inhibitor or nonspecific COX inhibitor is associated with significant short-term increase in spinal COX-2 expression.     

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.     

Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120

Astrocytes and microglia in the spinal cord have recently been reported to contribute to the development of peripheral inflammation-induced exaggerated pain states. Both lowering of thermal pain threshold (thermal hyperalgesia) and lowering of response threshold to light tactile stimuli (mechanical allodynia) have been reported. The notion that spinal cord glia are potential mediators of such effects is based on the disruption of these exaggerated pain states by drugs thought to preferentially affect glial function. Activation of astrocytes and microglia can release many of the same substances that are known to mediate thermal hyperalgesia and mechanical allodynia. The aim of the present series of studies was to determine whether exaggerated pain states could also be created in rats by direct, intraspinal immune activation of astrocytes and microglia. The immune stimulus used was peri-spinal (intrathecal, i.t.) application of the Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein, gp120. This portion of HIV-1 is known to bind to and activate microglia and astrocytes. Robust thermal hyperalgesia (tail-flick, TF, and Hargreaves tests) and mechanical allodynia (von Frey and touch-evoked agitation tests) were observed in response to i.t. gp120. Heat denaturing of the complex protein structure of gp120 blocked gp120-induced thermal hyperalgesia. Lastly, both thermal hyperalgesia and mechanical allodynia to i.t. gp120 were blocked by spinal pretreatment with drugs (fluorocitrate and CNI-1493) thought to preferentially disrupt glial function.     

The involvement of glia in long-term plasticity in the spinal dorsal horn of the rat

We have examined the potential role of spinal glial cells in the induction of C fiber-evoked long-term potentiation (LTP) in the spinal cord. Tetanic stimulation of the sciatic nerve induced longterm potentiation of C-fiber-evoked field potentials in the spinal dorsal horn in all rats. Following intrathecal fluorocitrate (1 nmol), a glial metabolic inhibitor, tetanic stimulation induced longterm depression (LTD) but not LTP. The effects of fluorocitrate were abolished by kynurenic acid or 2-amino-5-phosphonovaleric acid (AP-5), but not by 6,7-dinitroquinoxaline-2,3-dione (DNQX), picrotoxin or strychnine. These data suggest that spinal glial cells may modulate the central sensitization of nociceptive neurons via NMDA receptors.     

Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors

The relieving effects of electroacupuncture (EA) on mechanical allodynia and its mechanism related to the spinal opioid system were investigated in a rat model of neuropathic pain. To produce neuropathic pain in the tail, the right superior caudal trunk was resected between the S1 and S2 spinal nerves. Two weeks after the surgery, EA stimulation (2 or 100 Hz, 0.3 ms, 0.2-0.3 mA) was delivered to Zusanli (ST36) for 30 min. The degree of mechanical allodynia was evaluated quantitatively by touching the tail with von Frey hair (2.0 g) at 10 min intervals. These rats were then subjected to an i.t. injection with one of the three specific opioid agonists in successive ways: the mu agonist (DAMGO 25, 50 and 100 pmol), the delta agonist (DADELT II 0.5, 1 and 2 nmol), and the kappa agonist (U50488H 5, 10 and 20 nmol) separated by 10 min in cumulative doses. During 30 min of EA stimulation, specific opioid antagonists were subjected to i.t. injection: the mu antagonist (beta-FNA 5, 10 and 20 nmol), the delta antagonist (naltrindole 5, 10 and 20 nmol), and the kappa antagonist (nor-BNI 3, 6 and 12 nmol) separated by 10 min in cumulative doses. As a result, EA reduced the behavioral signs of mechanical allodynia. Two Hz EA induced a robust and longer lasting effect than 100 Hz. All three opioid agonists also showed relieving effects on mechanical allodynia. However, nor-BNI could not block the EA effects on mechanical allodynia, whereas beta-FNA or naltrindole significantly blocked EA effects. These results suggest that the mu and delta, but not kappa, opioid receptors in the spinal cord of the rat, play important roles in mediating relieving effects on mechanical allodynia induced by 2 Hz EA.     

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.