Spinal PGE Research Articles

Elevated Levels of PGE2-Metabolite in Cerebrospinal Fluid and Cox-2 Gene Polymorphisms in Patients with Chronic, Post Cholecystectomy Pain and Visceral Hyperalgesia Compared to Healthy Controls. A Hypothesis-Generating Pilot Study.

Chronic, abdominal pain remains a problem in a subset of patients after cholecystectomy. The cause is often obscure but central sensitization may be an important component and could theoretically be mediated by spinal PGE2, which is regulated by several cytokines. The aim of the study was to examine cerebrospinal fluid (CSF) of participants with post cholecystectomy syndrome and healthy volunteers for signs of PGE2 and cytokine mediated central sensitization. In phase 1 of the study, 83 subjects were included for DNA analysis, eight of these subjects with post cholecystectomy syndrome. We examined the SNPs rs5275, rs16944 and rs1800795 from the Cox-2, IL-1β and IL-6 genes respectively. In phase 2 of the study, we examined concentrations of PGE2-metabolite (PGEM), IL-1β and IL-6 in CSF and plasma from 6 patients with post cholecystectomy syndrome and visceral hyperalgesia and 11 pain free volunteers. We found a significant difference in distribution of the rs5275 SNP of the Cox-2 enzyme (CT-genotype=88% in pain group, 45% in pain free group, TT-genotype=0 in pain group, 41% in pain free group, p=0.05) but not in the other SNPs. PGEM, but not IL-6, was significantly elevated in CSF of the pain group (3.6 pg/mL, sd=1.9 vs 2.1 pg/mL, p=0.03), IL-1β was undetectable. We found elevated PGEM levels in CSF of patients with post cholecystectomy syndrome and visceral hyperalgesia, suggesting a central, possibly inflammatory component to the pain, and overrepresentation of the CT-genotype in the rs5275 SNP in the Cox2 gene, suggesting overexpression of Cox2 as a possible cause for elevated PGEM levels.

P2X7 receptor-mediated release of microglial prostanoids and miRNAs correlates with reversal of neuropathic hypersensitivity in rats.

P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1β, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.

The Central Analgesic Mechanism of YM-58483 in Attenuating Neuropathic Pain in Rats.

Calcium channel antagonists are commonly used to treat neuropathic pain. Their analgesic effects rely on inhibiting long-term potentiation, and neurotransmitters release in the spinal cord. Store-operated Ca(2+)channels (SOCCs) are highly Ca(2+)-selective cation channels broadly expressed in non-excitable cells and some excitable cells. Recent studies have shown that the potent inhibitor of SOCCs, YM-58483, has analgesic effects on neuropathic pain, but its mechanism is unclear. This experiment performed on spinal nerve ligation (SNL)-induced neuropathic pain model in rats tries to explore the mechanism, whereby YM-58483 attenuates neuropathic pain. The left L5 was ligated to produce the SNL neuropathic pain model in male Sprague-Dawley rats. The withdrawal threshold of rats was measured by the up-down method and Hargreaves' method before and after intrathecal administration of YM-58483 and vehicle. The SOCCs in the spinal dorsal horn were located by immunofluorescence. The expression of phosphorylated ERK and phosphorylated CREB, CD11b, and GFAP proteins in spinal level was tested by Western blot, while the release of proinflammatory cytokines (IL-1β, TNF-α, PGE2) was measured by enzyme-linked immunosorbent assay (ELISA). Intrathecal YM-58483 at the concentration of 300μM (1.5nmol) and 1000μM (10nmol) produced a significant central analgesic effect on the SNL rats, compared with control+vehicle (n=7, P<0.001). However, both could not prevent the development of neuropathic pain, compared with normal+saline (P<0.001). Immunofluorescent staining revealed that Orai1 and STIM1 (the two key components of SOCCs) were located in the spinal dorsal horn neurons. Western blot showed that YM-58483 could decrease the levels of P-ERK and P-CREB (n=10, #P<0.05), without affecting the expression of CD11b and GFAP (n=10, #P>0.05). YM-58483 also inhibited the release of spinal cord IL-1β, TNF-α, and PGE2, compared with control+vehicle (n=5, #P<0.001). The analgesic mechanism of YM-58483 may be via inhibiting central ERK/CREB signaling in the neurons and decreasing central IL-1β, TNF-α, and PGE2 release to reduce neuronal excitability in the spinal dorsal horn of the SNL rats.

Reduction of spinal PGE2 concentrations prevents swim stress-induced thermal hyperalgesia

We evaluated the association between spinal PGE2 and thermal hyperalgesia following repeated stress. Thermal nociception was determined in male Sprague–Dawley rats using the hot-plate test, before and after forced-swimming; non-conditioned rats served as controls. Animals were pretreated with ketoprofen or meloxicam, preferential COX-1 and COX-2 inhibitors, respectively. After the second hot-plate test, we measured serum corticosterone (stress marker), and lumbar spinal PGE2 (neuroinflammation marker) under peripheral inflammation (1% formalin plantar injection). Stressed rats displayed response latencies 40% shorter and inflammatory spinal PGE2 levels 95% higher than controls. Pretreatment with ketoprofen or meloxicam prevented hyperalgesia and elevation of spinal PGE2, increasing the escape behavior time during forced swimming 95% respect to saline-treated rats. Corticosterone levels in stressed rats were 97% higher than controls; COX inhibitors reduced them by 84%. PGE2 could participate in stress-induced hyperalgesia, learned helplessness, and corticosterone production, supporting the use of non-steroidal anti-inflammatory drugs (NSAIDs) for persistent pain associated with chronic stress and depression.

Activation of descending pain-facilitatory pathways from the rostral ventromedial medulla by cholecystokinin elicits release of prostaglandin-E 2 in the spinal cord

Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and “anti-opioid” by actions on pain-modulatory cells within the rostral ventromedial medulla (RVM). One consequence of activation of RVM CCK 2 receptors may be enhanced spinal nociceptive transmission; but how this might occur, especially in states of pathological pain, is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold after ligation of L 5/L 6 spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time-related increase in (prostaglandin-E 2) PGE 2 measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE 2 was approximately 5-fold and was observed at approximately 80 minutes post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-hydroxyindoleacetic acid (5-HIAA), a 5-hydoxytryptophan (5-HT) metabolite, as compared with controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT 3 antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE 2 and 5-HT in the spinal cord.

Spinal antinociceptive effects of cyclooxygenase inhibition during inflammation: Involvement of prostaglandins and endocannabinoids

Both cyclooxygenase-1 and -2 are expressed in the spinal cord, and the spinal COX product prostaglandin E 2 (PGE 2) contributes to the generation of central sensitization upon peripheral inflammation. Vice versa spinal COX inhibition is considered an important mechanism of antihyperalgesic pain treatment. Recently, however, COX-2 was shown to be also involved in the metabolism of endocannabinoids. Because endocannabinoids can have analgesic actions it is conceivable that inhibition of spinal COX produces analgesia not only by inhibition of PG synthesis but also by inhibition of endocannabinoid breakdown. In the present study, we recorded from spinal cord neurons with input from the inflamed knee joint and we measured the spinal release of PGE 2 and the endocannabinoid 2-arachidonoyl glycerol (2-AG) in vivo, using the same stimulation procedures. COX inhibitors were applied spinally. Selective COX-1, selective COX-2 and non-selective COX inhibitors attenuated the generation of spinal hyperexcitability when applied before and during development of inflammation but, when inflammation and spinal hyperexcitability were established, only selective COX-2 inhibitors reversed spinal hyperexcitability. During established inflammation all COX inhibitors reduced release of spinal PGE 2 almost equally but only the COX-2 inhibitor prevented breakdown of 2-AG. The reversal of spinal hyperexcitability by COX-2 inhibitors was prevented or partially reversed by AM-251, an antagonist at the cannabinoid-1 receptor. We conclude that inhibition of spinal COX-2 not only reduces PG production but also endocannabinoid breakdown and provide evidence that reversal of inflammation-evoked spinal hyperexcitability by COX-2 inhibitors is more related to endocannabinoidergic mechanisms than to inhibition of spinal PG synthesis.

Comparison of the effects of intrathecal administration of levobupivacaine and lidocaine on the prostaglandin E2 and glutamate increases in cerebrospinal fluid: a microdialysis study in freely moving rats

Bupivacaine has a lower incidence of transient neurological symptoms than lidocaine after intrathecal (i.t.) injection. The increased toxic potential of lidocaine does not support its use in the clinical setting and could be related to augmented levels of spinal prostaglandin E(2) (PGE(2)). We tested whether levobupivacaine leads to lower PGE(2) levels than lidocaine. Moreover, we compared the release of PGE(2) and glutamate after i.t. injections of levobupivacaine or lidocaine. Rats were anaesthetized for implantation of an i.t. dialysis catheter. This allowed sampling dialysates of cerebrospinal fluid (CSF) for measuring PGE(2) and glutamate levels. The microdialysis setting included baseline sampling and was followed by an i.t. injection of levobupivacaine 250 microg, 100 microg, or saline. PGE(2) and glutamate levels in CSF were analysed for 4 h. In addition, the residual effect of a second i.t. injection on, respectively, of PGE(2) and glutamate changes was compared after injection of either 250 or 100 microg levobupivacaine, 1000 or 400 microg lidocaine, or saline. Prolonged spinal PGE(2) increases lasting 50-120 min were observed after levobupivacaine injection. Higher PGE(2) concentrations were observed after the second lidocaine 1000 microg injection. Glutamate release after the second injection did not vary between the local anaesthetic groups. Spinal PGE(2) levels are similarly increased after i.t. levobupivacaine injection of 250 and 100 microg. A higher PGE(2) response was observed after a second i.t. injection in the animals receiving 1000 microg lidocaine than those receiving 400 mg lidocaine or either dose of levobupivacaine.

A selective role for α3 subunit glycine receptors in inflammatory pain

GlyR α3 has previously been found to play a critical role in pain hypersensitivity following spinal PGE2 injection, complete Freund's adjuvant (CFA) and zymosan induced peripheral inflammation. In this study, although all models displayed typical phenotypic behaviours, no significant differences were observed when comparing the pain behaviours of Glra3−/− and wild-type littermates following the injection of capsaicin, carrageenan, kaolin/carrageenan or monosodium iodoacetate, models of rheumatoid and osteoarthritis, respectively. However, clear differences were observed following CFA injection (p < 0.01). No significant differences were observed in the pain behaviours of Glra3−/− and wild-type littermates following experimentally induced neuropathic pain (partial sciatic nerve ligation). Similarly, Glra3−/− and wild-type littermates displayed indistinguishable visceromotor responses to colorectal distension (a model of visceral pain) and in vivo spinal cord dorsal horn electrophysiology revealed no differences in responses to multimodal suprathreshold stimuli, intensities which equate to higher pain scores such as those reported in the clinic. These data suggest that apart from its clear role in CFA- and zymosan-induced pain sensitisation, hypersensitivity associated with other models of inflammation, neuropathy and visceral disturbances involves mechanisms other than the EP2 receptor – GlyR α3 pathway.

Acetaminophen prevents hyperalgesia in central pain cascade

Acetaminophen is an analgesic and antipyretic drug believed to exert its effect through interruption of nociceptive processing. In order to determine whether this effect is due to peripheral or central activity, we studied the efficacy of systemic (oral) and intrathecal (IT) application of acetaminophen in preventing the development of hyperalgesia induced through the direct activation of pro-algogenic spinal receptors. Spinal administration of substance P (SP, 30 nmol, IT) in rats produced a decreased thermal threshold, indicating centrally mediated hyperalgesia. Pretreatment of rats with oral acetaminophen (300 mg/kg), but not vehicle, significantly attenuated IT SP-induced hyperalgesia. Acetaminophen given IT also produced a dose-dependent (10-200 microg) antinociceptive effect. In addition, oral acetaminophen suppressed spinal PGE(2) release evoked by IT SP in an in vivo IT dialysis model. The ability of IT as well as oral acetaminophen to reverse this spinally initiated hyperalgesia emphasizes the likely central action and bioavailability of the systemically delivered drug. Jointly, these data argue for an important central antihyperalgesic action of acetaminophen.

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)-α, interleukin (IL)-1β, 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-1β, IL-6, and TNF-α 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-1β, IL-6, or TNF-α. 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.

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES‐1‐deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.

Peripheral inflammation modifies the effect of intrathecal IL-1β on spinal PGE 2 production mainly through cyclooxygenase-2 activity. A spinal microdialysis study in freely moving rats

Acute inflammation induces upregulation of IL-1β both at the site of the peripheral inflammation and in the cerebrospinal fluid (CSF). The central increase of IL-1β mainly contributes to the development of hypersensitivity. However, the spinal mechanisms for the effects of IL-1β in nociceptive transmission are incompletely understood. It is also unknown whether previous sensitization changes IL-1β activity. We therefore investigated the dose–effect relationship of intrathecal (i.t.) IL-1β on spinal PGE 2 production in the absence and presence of peripheral formalin inflammation with spinal microdialysis in freely moving rats. The possible involvement of cyclooxygenase (COX) isoforms in the IL-1β-mediated spinal PGE 2 production on the background of peripheral formalin inflammation was further evaluated with the selective COX-1 and COX-2 inhibitors. We found that the i.t. administration of IL-1β, with doses of 1, 2, 8, or 16 ng, increased PGE 2 levels in CSF in a dose-related fashion. This IL-1β-evoked PGE 2 release occurred within 30 min after IL-1β administration, peaked at 30–60 min interval, and returned gradually to the baseline level within 4 h. Peripheral formalin inflammation in the paw induced a more prolonged effect of spinal IL-1β with larger PGE 2 releases in the CSF compared with the non-inflammatory state, suggesting that peripheral inflammation enhances central sensitization. The COX-2 inhibitor SC58236 (15 mg/kg) reduced the IL-1β-mediated PGE 2 increase in CSF by 86% while the COX-1 inhibitor SC58560 (15 mg/kg) had less effect (28%). Our study suggests that mainly the COX-2 enzyme mediates the IL-1β-induced increase in spinal PGE 2 in the presence of peripheral formalin inflammation.

HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain.

BackgroundTo examine the role of inflammatory mediators in neuropathic pain, we used a replication-defective genomic herpes simplex virus (HSV)-based vector containing the coding sequence for the anti-inflammatory peptide interleukin (IL)-4 under the transcriptional control of the HSV ICP4 immediate early promoter, vector S4IL4, to express IL-4 in dorsal root ganglion (DRG) neurons in vivo.ResultsSubcutaneous inoculation of S4IL4 in the foot transduced lumbar DRG to produce IL-4. Transgene-mediated expression of IL-4 did not alter thermal latency or tactile threshold in normal animals, but inoculation of S4IL4 1 week after spinal nerve ligation (SNL) reduced mechanical allodynia and reversed thermal hyperalgesia resulting from SNL. Inoculation of S4IL4 1 week before SNL delayed the development of thermal hyperalgesia and tactile allodynia, but did not prevent the ultimate development of these manifestations of neuropathic pain. S4IL4 inoculation suppressed non-noxious-induced expression of c-Fos immunoreactivity in dorsal horn of spinal cord and reversed the upregulation of spinal IL-1β, PGE2, and phosphorylated-p38 MAP kinase, characteristic of neuropathic pain.ConclusionHSV-mediated expression of IL-4 effectively reduces the behavioral manifestations of neuropathic pain, and reverses some of the biochemical and histologic correlates of neuropathic pain at the spinal level.

Mechanisms of antinociception of spinal galanin: how does galanin inhibit spinal sensitization?

Galanin by a spinal action has been shown to have an antihyperalgesic action. Thus, in rats with lumbar intrathecal (IT) catheters, the thermal hyperalgesia evoked by carrageenan paw injection was blocked by IT delivery of galanin(1-29) (Gal(1-29)) and galanin(2-11) (Gal(2-11)) with the rank order of activity being Gal(1-29)>Gal(2-11). We sought to determine whether this spinal action reflects an effect upon afferent transmitter release, e.g., substance P (SP), and/or on secondary neurons, e.g., signaling postsynaptic to neurokinin 1 (NK1) receptor activation. To address the question on afferent release, we investigated the effect of IT administration of galanin on tissue injury-induced spinal NK1 internalization (an indicator of SP release). Noxious stimulation (paw compression) produced an increase in NK1 internalization in dorsal horn lamina I. IT pretreatment of rats with Gal(1-29) and Gal(2-11) significantly attenuated the evoked NK1 internalization, with the rank order of activity being Gal(1-29)>Gal(2-11)>saline. To address the question of postsynaptic action, we examined the effects of IT galanin upon IT SP-induced thermal hyperalgesia and spinal PGE2 release. Application of SP (30 nmol) directly to spinal cord led to a decrease in thermal thresholds and a profound increase in PGE(2) concentration in spinal dialysates. Both phenomena were reversed by Gal(1-29) and Gal(2-11) (10nmol, IT). These findings suggest that the antihyperalgesic effect of spinal galanin is due to its action on sites both presynaptic (inhibition of SP release) and postsynaptic (blockade of SP-evoked hyperalgesia and PGE2 production) to the primary afferents.

GlyR alpha3: an essential target for spinal PGE2-mediated inflammatory pain sensitization.

Prostaglandin E2 (PGE2) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR alpha3) by PGE2-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR alpha3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR alpha3 not only lack the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation. Thus, GlyR alpha3 may provide a previously unrecognized molecular target in pain therapy.

Role of prostaglandin receptor subtype EP 1 in prostaglandin E 2-induced nociceptive transmission in the rat spinal dorsal horn

It has been indicated that prostaglandin E 2 (PGE 2) and the receptor for PGE 2 (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE 2 and EP 1 receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca 2+] 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 EP 1 antagonist, 6-[(2 S,3 S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5 Z-hexenoic acid (ONO-8711), on the intrathecal PGE 2-induced hyperalgesia was examined. In [Ca 2+] i assays, we measured [Ca 2+] i in the dorsal horn of spinal cord slices and examined the effects of PGE 2 and ONO-8711 perfusion on the [Ca 2+] i changes. In situ hybridization using EP 1 digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L 4 and L 5 dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE 2 administration. Intrathecal administration of ONO-8711 attenuated the PGE 2-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE 2-induced [Ca 2+] i increment in laminae II–VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP 1 hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE 2 activates the EP 1 receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca 2+] i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE 2-induced nociceptive transmission.

Novel therapeutic agents: Gene transfer of interleukin-4 (IL-4) produces persistent antinociception and suppresses the induction of spinal PGE2 in neuropathic pain

Inflammatory cytokines (e.g. TNF alpha, IL-1 beta and IL-6) and prostaglandin E2 in spinal cord appear to play a role in the neuropathic pain. Interleukin-4 (IL-4) is an anti-inflammatory cytokine that inhibits production of TNF alpha, IL-1 and IL-6, among other actions. In the present study, we examined the effect of herpes simplex virus (HSV)-based vector-mediated transfer of the gene coding for IL-4 (vector S4IL4) to lumbar DRG in rats with neuropathic pain. Primary DRG neurons in culture transduced with S4IL4 released IL-4 into the medium measured by ELISA. Subcutaneous inoculation of S4IL4 into the paw resulted in expression of IL-4 in L4-6 DRG and sciatic nerve in vivo detected by ELISA. Mechanical threshold was determined by stimulation of the plantar surface of the hindpaw with graded von Frey monofilaments; the threshold was established using the up-down method (Dixon). Thermal latency was assessed by thermal evoked paw withdrawal. In normal rats, neither vector expressing IL-4 nor control vector changed mechanical threshold or thermal latency. Selective L5 spinal nerve ligation (SNL) resulted in neuropathic pain characterized by mechanical allodynia and thermal hyperalgesia. Subcutaneous inoculation of vector S4IL4 produced a significant antiallodynic effect and reversed thermal hyperalgesia. Inoculation with a control HSV vector carrying the lacZ reporter transgene in the tk locus (vector SHZ) did not alter either mechanical threshold or thermal latency. Inoculation of S4IL4 one week prior to SNL delayed development of tactile allodynia and thermal hyperalgesia. Inoculation of S4IL4 suppressed gentle touch-induced expression of c-fos immunoreactivity in spinal cord. Inoculation of S4IL4 suppressed upregulation of spinal IL-1 beta and prostaglandin E2 in rats with neuropathic pain. The results demonstrate that recombinant herpes virus expressing IL-4 may be antinociceptive in the neuropathic pain.

Mechanisms of pain in arthritis.

Inflammation in the joint causes peripheral sensitization (increase of sensitivity of nociceptive primary afferent neurons) and central sensitization (hyperexcitability of nociceptive neurons in the central nervous system). The processes of sensitization are thought to be the basis of arthritic pain that appears as spontaneous pain (joints at rest) and hyperalgesia (augmented pain response on noxious stimulation and pain on normally nonpainful stimulation). Sensitization also facilitates efferent neuronal processes through which the nervous system influences the inflammatory process. Peripheral sensitization is produced by the action of inflammatory mediators such as bradykinin, prostaglandins, neuropeptides, and cytokines which activate corresponding receptors in proportions of nerve fibers. In addition, the expression of receptors, for example, bradykinin and neurokinin 1 receptors, is upregulated during inflammation. The development of hyperexcitability of spinal cord neurons is produced by various transmitter/receptor systems that constitute and modulate synaptic activation of the neurons. The key transmitter is glutamate that activates N-methyl-d-aspartate (NMDA) and non-NMDA receptors on spinal cord neurons. Blockade of these receptors prevents and reduces central sensitization. Excitatory neuropeptides (substance P and calcitonin gene-related peptide) further central sensitization. Central sensitization also is facilitated by mediators that have complex actions (e.g., prostaglandin E(2)). Spinal PGE(2) binds to receptors at presynaptic endings of primary afferent neurons (thus influencing synaptic release) and to receptors on postsynaptic spinal cord neurons. The administration of PGE(2) to the spinal cord surface produces changes of responsiveness of spinal neurons similar to peripheral inflammation, and spinal indomethacin to the spinal cord attenuates development of hyperexcitability significantly.

The role of spinal neurokinin-1 and glutamate receptors in hyperalgesia and allodynia induced by prostaglandin E 2 or zymosan in the rat

Recent research has focused on prostaglandins in the central nervous system and their contribution to hyperalgesia and allodynia. This study sought to establish whether neurokinin-1 (NK-1) receptors and glutamate receptors are involved in the hyperalgesic and allodynic effects of spinally administered prostaglandin E 2 (PGE 2) in rats, and also to determine if the same receptors are involved the hyperalgesia induced by intraplantar administration of zymosan, an inflammatory agent which is known to evoke spinal PGE 2 release. Spinal application of antagonists of the NK-1 receptor, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate or metabotropic glutamate receptor significantly attenuated the decrease in mechanical paw withdrawal response thresholds produced by either spinal administration of PGE 2 or intraplantar administration of zymosan. The decrease in thermal paw withdrawal response latencies induced by PGE 2, but not by zymosan, was significantly attenuated by spinal administration of an N-methyl- d-aspartate (NMDA) receptor antagonist, an AMPA/kainate receptor antagonist, or a metabotropic glutamate receptor antagonist. Allodynia induced by PGE 2 was significantly alleviated by antagonists of NMDA or AMPA/kainate receptors. These results suggest that both PGE 2-induced and zymosan-induced mechanical hyperalgesia are mediated in part through activation of NK-1, AMPA/kainate and metabotropic glutamate receptors. PGE 2-induced, but not zymosan-induced, thermal hyperalgesia is mediated in part by activation of NMDA, AMPA/kainate and metabotropic glutamate receptors. Activation of both NMDA and AMPA/kainate receptors contribute to PGE 2-induced allodynia.

Nociceptin and dynorphin A(1-17) produce antianalgesia through independent systems in mice

The administration of dynorphin A(1-17), Dyn, intrathecally (i.t.) or of nociceptin, intracerebroventricularly (i.c.v.) produces antianalgesic actions against i.t. morphine in the tail flick test in mice. The antianalgesic action of nociceptin is mediated by spinal PGE 2 and attenuated by i.t. PGD 2 or indomethacin. The Dyn response is mediated by release of IL 1β in the spinal cord to activate an ascending pathway to the brain and in turn releases IL 1β in the brain which activates a descending pathway to the spinal cord. The present work investigated the possibility that the action of IL 1β in the Dyn system might release prostaglandins so that the Dyn and nociceptin antianalgesic systems would overlap at these points. The results indicated that in the Dyn system neither the IL 1β in the spinal cord or brain implicated prostaglandin release because i.t. and i.c.v. PGD 2 and indomethacin did not affect Dyn-induced antianalgesia. In addition, nociceptin-induced antianalgesia did not involve components in the Dyn system. Thus, the Dyn and nociceptin antianalgesic systems did not overlap and each were independent systems.