Effects of selective COX-1 and -2 inhibition on formalin-evoked nociceptive behaviour and prostaglandin E(2) release in the spinal cord.

Nociception evoked prostaglandin (PG) release in the spinal cord considerably contributes to the induction of hyperalgesia and allodynia. To evaluate the relative contribution of cyclooxygenase-1 (COX-1) and COX-2 in this process we assessed the effects of the selective COX-1 inhibitor SC560 and the selective COX-2 inhibitor celecoxib on formalin-evoked nociceptive behaviour and spinal PGE(2) release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin-evoked PGE(2) raise. In contrast, celecoxib (10 and 20 mg/kg) was ineffective in both regards, i.e. the flinching behaviour was largely unaltered and the formalin-induced PGE(2) raise as assessed using microdialysis was only slightly, not significantly reduced. This suggests that the formalin-evoked rapid PG release was primarily caused by COX-1 and was independent of COX-2. Mean free spinal cord concentrations of celecoxib during the formalin assay were 32.0 +/- 4.5 nM, thus considerably higher than the reported IC50 for COX-2 (3-7 nM). Therefore, the lack of efficacy of celecoxib is most likely not to be a result of poor tissue distribution. COX-2 mRNA and protein expression in the spinal cord were not affected by microdialysis alone but the mRNA rapidly increased following formalin injection and reached a maximum at 2 h. COX-2 protein was unaltered up to 4 h after formalin injection. The time course of COX-2 up-regulation suggests that the formalin-induced nociceptive response precedes COX-2 protein de novo synthesis and may therefore be unresponsive to COX-2 inhibition. Considering the results obtained with the formalin model it may be hypothesized that the efficacy of celecoxib in early injury evoked pain may be less than that of unselective NSAIDs.     

Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Ethyl Pyruvate Attenuates Formalin-induced Inflammatory Nociception by Inhibiting Neuronal ERK Phosphorylation

ABSTRACT: BACKGROUND: Ethyl pyruvate (EP) possesses anti-inflammatory activity. However, the potential anti-nociceptive value of EP for the treatment of the inflammatory nociception is largely unknown. We investigated whether EP could have any anti-nociceptive effect on inflammatory pain, after systemic administration of EP (10, 50, and 100 mg/kg, i.p.), 1 hour before formalin (5%, 50 mul) injection into the plantar surface of the hind paws of rats. RESULTS: EP significantly decreased formalin-induced nociceptive behavior during phase II, the magnitude of paw edema, and the activation of c-Fos in L4-L5 spinal dorsal horn. EP also attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) in the neurons of L4-L5 spinal dorsal horn after formalin injection. Interestingly, the i.t. administration of PD-98059, an ERK upstream kinase (MEK) inhibitor, completely blocked the formalin-induced inflammatory nociceptive responses. CONCLUSIONS: These results demonstrate that EP may effectively inhibit formalin-induced inflammatory nociception via the inhibition of neuronal ERK phosphorylation in the spinal dorsal horn, indicating its therapeutic potential in suppressing acute inflammatory pain.     

MK-801抑制福尔马林实验引起的大鼠脊髓背角环氧合酶-2的表达

应用免疫组织化学方法,观察鞘内注射N-methyl—D—aspartate(NMDA)受体拮抗剂MK-801对福尔马林实验引起的大鼠脊髓背角环氧合酶-2(cyclooxygenase-2,COX-2)表达的影响。结果表明：MK-801对福尔马林实验引起的第1相缩足反射仅有一定抑制作用,但对第2相缩足反射有显著的抑制作用,且呈剂量依赖性。与这种行为学的变化相对应,MK-801可显著抑制福尔马林实验引起的脊髓背角COX-2表达的增加,并且这种抑制作用与MK-801的剂量呈正相关。这些结果表明,在福尔马林实验中,NMDA受体的活动是引起脊髓背角COX-2表达增加的原因之一。     

Anesthesiologists' practice patterns for treatment of postoperative nausea and vomiting in the ambulatory Post Anesthesia Care Unit

Background

Induction of the COX-2 isoenzyme appears to play a major role in the genesis of central sensitization after nociceptive stimulation. This study aimed to investigate the efficacy of a single, oral dose of the specific COX-2 inhibitor-valdecoxib in attenuating the central sensitization – induced secondary hyperalgesia in a heat/capsaicin pain model in healthy volunteers.

Methods

The study was a randomized, double blind, placebo controlled, crossover, single dose efficacy trial using 20 healthy volunteers. Two hours following placebo or 40 mg, PO valdecoxib, participants underwent skin sensitization with heat/capsaicin, as well as supra-threshold pain and re-kindling measurements according to an established, validated pain model. Subjects rated pain intensity and unpleasantness on a visual analog scale and the area of secondary hyperalgesia was serially mapped.

Results

The area of secondary hyperalgesia produced after 40 mg of valdecoxib was no different than that after placebo. Furthermore, there were no significantly relevant differences when volunteers were treated with valdecoxib or placebo in relation to either cold- or hot pain threshold or the intensity of pain after supra-threshold, thermal pain stimulation.

Conclusion

We demonstrated that a single, oral dose of valdecoxib when does not attenuate secondary hyperalgesia induced by heat/capsaicin in a cutaneous sensitization pain model in healthy volunteers.     

COX-2 expression and function in the hyperalgesic response to paw inflammation in mice

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.     

Spinal p38beta isoform mediates tissue injury-induced hyperalgesia and spinal sensitization

Antagonist studies show that spinal p38 mitogen-activated protein kinase plays a crucial role in spinal sensitization. However, there are two p38 isoforms found in spinal cord and the relative contribution of these two to hyperalgesia is not known. Here we demonstrate that the isoforms are distinctly expressed in spinal dorsal horn: p38alpha in neurons and p38beta in microglia. In lieu of isoform selective inhibitors, we examined the functional role of these two individual isoforms in nociception by using intrathecal isoform-specific antisense oligonucleotides to selectively block the expression of the respective isoform. In these rats, down-regulation of spinal p38beta, but not p38alpha, prevented nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia induced by activation of spinal neurokinin-1 receptors through intrathecal injection of substance P. Both intraplantar formalin and intrathecal substance P produced an increase in spinal p38 phosphorylation and this phosphorylation (activation) was prevented when spinal p38beta, but not p38alpha, was down-regulated. Thus, spinal p38beta, probably in microglia, plays a significant role in spinal nociceptive processing and represents a potential target for pain therapy.     

Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing

We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.     

COX-2 expression and function in the hyperalgesic response to paw inflammation in mice

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2^−/− mice is similar to that of their Cox-2^+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2^−/− versus Cox-2^+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.     

Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.     

Comparison of the antinociceptive effects of ibuprofen arginate and ibuprofen in rat models of inflammatory and neuropathic pain

AimsIbuprofen arginate is a highly soluble salt formed by combining racemic ibuprofen with the amino acid l-arginine. This formulation is absorbed faster, and it is safe and effective in treating many forms of mild to moderate pain. We compared the analgesic effect of ibuprofen arginate and conventional ibuprofen in rat models of pain.Main methodsMechanical and cold allodynia were assessed in the chronic constriction injury (CCI) model of neuropathic pain, and mechanical allodynia was also examined in capsaicin-injected rats (a model of central sensitization). Inflammatory hypersensitivity was assessed with the formalin test. Ibuprofen-l-arginine, ibuprofen, l-arginine or saline was administered orally on a daily basis after CCI or capsaicin injection, and the von Frey and cold plate tests were performed on days 1, 3 and 7 after CCI or capsaicin administration. In the formalin-induced inflammatory pain test, the drugs were administered 30 min before formalin injection.Key findingsIbuprofen only exerts an antinociceptive effect in the formalin model whereas ibuprofen-l-arginine exerts antinociceptive effects on both mechanical and cold allodynia induced by CCI, mechanical allodynia induced by capsaicin injection, and in phase 2 of the formalin test, exhibiting superior antinociceptive activity to ibuprofen in all these tests. l-Arginine only exerted antinociceptive effects on cold allodynia in CCI.SignificanceThese results demonstrate that ibuprofen arginate has stronger antinociceptive effects than ibuprofen in all the models used, suggesting it might improve the therapeutic management of neuropathic and inflammatory pain.     

Antinociception produced by systemic, spinal and supraspinal administration of amiloride in mice.

This study investigates the antinociceptive and antihyperalgesic action caused by i.p., i.t. or i.c.v. injections of amiloride when assessed against formalin, capsaicin-induced licking, acetic acid-induced writhing and glutamate-induced hyperalgesia in mice. The systemic, spinal and supraspinal administration of amiloride causes dose-related antinociception when assessed against acetic acid-induced writhing, formalin and capsaicin-induced licking. In addition, amiloride administered by the same routes produced graded inhibition of glutamate-induced hyperalgesia in mice. Together, these results suggest, that amiloride or its derivatives may constitute a strategy for the development of new antinociceptive drugs.     

A comparison of the glutamate release inhibition and anti-allodynic effects of gabapentin, lamotrigine, and riluzole in a model of neuropathic pain

The effects of treatment with the anti-convulsant agents, lamotrigine and riluzole were compared with gabapentin in a rat experimental model of neuropathic pain. Rats were treated intraperitoneally, with gabapentin (30, 100 and 300 mg/kg), lamotrigine (2, 10 and 50 mg/kg) or riluzole (6 and 12 mg/kg) prior to, and every 12 h for 4 days following chronic constriction injury (CCI) of the sciatic nerve. Mechanical and cold sensitivity were assessed prior to surgery (baseline) and then at 4, 8 and 12 days following CCI. The four-day treatment with each of the agents was effective at producing reductions in the development of mechanical and cold hypersensitivity for periods ranging from the fourth to 12th day. The highest doses of each of the agents were also assessed on formalin-induced nociceptive behaviors and on formalin-induced increases in extracellular glutamate (Glu) and aspartate (Asp) in the spinal cord dorsal horn (SCDH) of awake behaving rats using in vivo microdialysis. Nociceptive scores in formalin test were significantly decreased by gabapentin (300 mg/kg i.p.) and riluzole (12 mg/kg i.p.), but not by lamotrigine (50 mg/kg i.p.). Formalin-induced increases in glutamate levels in SCDH were lowered significantly, as compared with the controls, with all drugs both in the first phase and second phases, with the greatest effects for riluzole and gabapentin. Similar suppressive effects of the drugs were observed on formalin-induced increases in spinal aspartate, except that gabapentin and lamotrigine produced effects only during the second phase. Riluzole produced profound and prolonged reductions in the spinal levels of glutamate and aspartate both for basal and formalin-stimulated release. In conclusion, the results suggest that the anti-convulsant agents gabapentin, lamotrigine and riluzole may reduce the development of hyperalgesia in a rat model of neuropathic pain by reducing the spinal release of glutamate. Riluzole's pronounced suppressive effects on spinal EAA levels is attributed to its established role as a glutamate release inhibitor and an enhancer of glutamate transporter activity.     

Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways.     

Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases

Background

Numerous studies have implicated spinal extracellular signal-regulated kinases (ERKs) as mediators of nociceptive plasticity. These studies have utilized pharmacological inhibition of MEK to demonstrate a role for ERK signaling in pain, but this approach cannot distinguish between effects of ERK in neuronal and non-neuronal cells. The present studies were undertaken to test the specific role of neuronal ERK in formalin-induced inflammatory pain. Dominant negative MEK (DN MEK) mutant mice in which MEK function is suppressed exclusively in neurons were tested in the formalin model of inflammatory pain.

Results

Formalin-induced second phase spontaneous pain behaviors as well as thermal hyperalgesia measured 1 – 3 hours post-formalin were significantly reduced in the DN MEK mice when compared to their wild type littermate controls. In addition, spinal ERK phosphorylation following formalin injection was significantly reduced in the DN MEK mice. This was not due to a reduction of the number of unmyelinated fibers in the periphery, since these were almost double the number observed in wild type controls. Further examination of the effects of suppression of MEK function on a downstream target of ERK phosphorylation, the A-type potassium channel, showed that the ERK-dependent modulation of the A-type currents is significantly reduced in neurons from DN MEK mice compared to littermate wild type controls.

Conclusion

Our results demonstrate that the neuronal MEK-ERK pathway is indeed an important intracellular cascade that is associated with formalin-induced inflammatory pain and thermal hyperalgesia.     

Antipruritic and antihyperalgesic actions of loperamide and analogs

Loperamide and three of its analogs were evaluated for their ability to inhibit binding to cloned human opioid receptor subtypes and to produce antipruritis and antinociception following local s.c. administration to rodents. All four compounds were fully efficacious agonists with affinities of 2 to 4 nM for the cloned human mu opioid receptor. Local s.c. injection of loperamide, ADL 01-0001 or ADL 01-0002 at the same site as the introduction of the pruritogenic compound 48/80 resulted in antipruritic activity in a mouse model of itch. Similarly, i.paw or i.pl. administration of compounds ADL 01-0001, ADL 01-0002 and ADL 01-0003 to inflamed paws caused potent antinociception, inhibiting late phase formalin-induced flinching, Freund's adjuvant-induced mechanical hyperalgesia and tape stripping-induced mechanical hyperalgesia. Loperamide and its analogs were efficacious in animal models of itch and inflammatory pain, and may have potential therapeutic utility as antipruritic and antihyperalgesic agents.     

Effects of the selective sigma‐1 receptor antagonist S1RA on formalin‐induced pain behavior and neurotransmitter release in the spinal cord in rats

We have previously shown that the selective sigma‐1 receptor (σ₁R) antagonist S1RA (E‐52862) inhibits neuropathic pain and activity‐induced spinal sensitization in various pre‐clinical pain models. In this study we characterized both the behavioral and the spinal neurochemical effects of S1RA in the rat formalin test. Systemic administration of S1RA produced a dose‐related attenuation of flinching and lifting/licking behaviors in the formalin test. Neurochemical studies using concentric microdialysis in the ipsilateral dorsal horn of awake, freely moving rats revealed that the systemic S1RA‐induced antinociceptive effect occurs concomitantly with an enhancement of noradrenaline levels and an attenuation of formalin‐evoked glutamate release in the spinal dorsal horn. Intrathecal pre‐treatment with idazoxan prevented the systemic S1RA antinociceptive effect, suggesting that the S1RA antinociception depends on the activation of spinal α₂‐adrenoceptors which, in turn, could induce an inhibition of formalin‐evoked glutamate release. When administered locally, intrathecal S1RA inhibited only the flinching behavior, whereas intracerebroventricularly or intraplantarly injected also attenuated the lifting/licking behavior. These results suggest that S1RA supraspinally activates the descending noradrenergic pain inhibitory system, which may explain part of its antinociceptive properties in the formalin test; however, effects at other central and peripheral sites also account for the overall effect.

     

Fluorocitrate,an Inhibitor of Glial Metabolism,Inhibits the Up-Regulation of NOS Expression,Activity and NO Production in the Spinal Cord Induced by Formalin Test in Rats

Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague–Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I–II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord. X.-C. Sun, W.-N. Chen and S.-Q. Li contributed equally to this work.