R-flurbiprofen reduces neuropathic pain in rodents by restoring endogenous cannabinoids.

BACKGROUND: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPARgamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. CONCLUSION: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.

Cofilin phosphorylation is involved in nitric oxide/cGMP-mediated nociception.

There is convincing evidence that nitric oxide (NO), cGMP and cGMP-dependent protein kinase I (PKG-I) are involved in the development of hyperalgesia in response to noxious stimuli. However, downstream target proteins contributing to nociception have not been completely identified so far. Several reports indicate a role of the NO/cGMP/PKG cascade in the regulation of neurite outgrowth which is suggested to be involved in specific mechanisms of nociception. Since neurite outgrowth is strongly dependent on modulation of cytoskeleton proteins we were interested in the impact of PKG-I activation on the actin cytoskeleton and its role in inflammatory hyperalgesia. Therefore we investigated the actin-destabilising protein cofilin and its NO-dependent effects in vitro in primary neuronal cultures as well as in vivo in the zymosan-induced paw inflammation model in rats. In primary neurons from rats, treatment with the PKG-I activator 8-Br-cGMP induced a time-dependent phosphorylation of cofilin and significantly increased neurite outgrowth. Further functional analysis revealed that the underlying signal transduction pathways involve activation of the Rho-GTPases RhoA, Rac1 and Cdc42 and their corresponding downstream targets Rho-kinase (ROCK) and p21-activated kinase (PAK). In vivo, treatment of rats with the NO-synthase inhibitor l-NAME and the ROCK-inhibitor Y-27632, respectively, led to a significant decrease of cofilin phosphorylation in the spinal cord and resulted in antinociceptive effects in a model of inflammatory hyperalgesia. Our results suggest that cofilin represents a downstream target of NO/cGMP/PKG signal transduction in neurons thus indicating that it is involved in NO-mediated nociception.

Antinociceptive activity of the S1P-receptor agonist FTY720.

FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.

Low-dose methotrexate reduces peripheral nerve injury-evoked spinal microglial activation and neuropathic pain behavior in rats.

Peripheral nerve injuries that provoke neuropathic pain are associated with microglial activation in the spinal cord. We have investigated the characteristics of spinal microglial activation in three distinct models of peripheral neuropathic pain in the rat: spared nerve injury (SNI), chronic constriction injury, and spinal nerve ligation. In all models, dense clusters of cells immunoreactive for the microglial marker CD11b formed in the ipsilateral dorsal horn 7 days after injury. Microglial expression of ionised calcium binding adapter molecule 1 (Iba1) increased by up to 40% and phosphorylation of p38 mitogen-activated protein kinase, a marker of microglial activity, by 45%. Expression of the lysosomal ED1-antigen indicated phagocytic activity of the cells. Unlike the peripheral nerve lesions, rhizotomy produced only a weak microglial reaction within the spinal gray matter but a strong activation of microglia and phagocytes in the dorsal funiculus at lumbar and thoracic spinal cord levels. This suggests that although degeneration of central terminals is sufficient to elicit microglial activation, it does not account for the inflammatory response in the dorsal horn after peripheral nerve injury. Early intrathecal treatment with low-dose methotrexate, beginning at the time of injury, decreased microglial activation, reduced p38 phosphorylation, and attenuated pain-like behavior after SNI. In contrast, systemic or intrathecal delivery of the glucocorticoid dexamethasone did not inhibit the activation of microglia or reduce pain-like behavior. We confirm that microglial activation is crucial for the development of pain after nerve injury, and demonstrates that suppression of this cellular immune response is a promising approach for preventing neuropathic pain.

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors.

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

The effects of COX-2 selective and non-selective NSAIDs on the initiation and progression of atherosclerosis in ApoE-/- mice.

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE(-/-) mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined. The results showed that neither the COX-2 selective inhibitors nor naproxen had a significant impact on the initiation and progression of atherosclerosis in diet-fed ApoE(-/-) mice, although both celecoxib and rofecoxib showed a tendency to reduce plaque size. This slight effect may be due to selective inhibition of COX-2 activity because the COX-2 expression was not altered in the plaque. Plasma lipid levels were also not significantly influenced by these drugs. Interestingly, in ApoE(-/-) mice that have been fed with normal chow, we found an increased incidence of plaque formation after treatment with celecoxib and rofecoxib, indicating that coxibs may promote the initiation of atherosclerosis. This effect was probably masked in diet-fed mice by the more pronounced effects of the high cholesterol diet. In conclusion, the reduction in diet-induced plaque size in animals fed a high fat diet and the promotion of atherosclerosis in mice on a normal diet indicate a dual role of the coxibs. In advanced stages of atherosclerosis, they may exert antithrombotic properties due to their COX-2 inhibiting activity, whereas in very early stages they may favor the initiation of atherogenesis. However, because these results were only observed in ApoE(-/-) and not in wild-type animals, coxibs may increase the risk of thrombosis in patients with a predisposition for thrombotic complications.

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury-evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

The glutamate transporter GLAST is involved in spinal nociceptive processing.

GLAST and GLT-1 are the most abundant glutamate transporters in the CNS and protect neurons from glutamate neurotoxicity. Here, we investigated the role of GLAST in spinal nociceptive processing. GLAST protein expression was not altered after treatment of rats with either formalin or zymosan. Surprisingly, knock-down of GLAST in the spinal cord using antisense-oligonucleotides decreased glutamate concentrations in cerebrospinal fluid (CSF) and reduced the nociceptive behaviour in the rat formalin assay. However, it did not influence thermal hyperalgesia in the zymosan-induced paw inflammation model indicating that GLAST is associated with spontaneous rather than inflammatory nociception. Mechanisms that might explain the decreased response in the formalin assay may include compensatory activation of other glutamate transporters, inhibition of glutamate release or disturbance of glutamate recycling. In conclusion, these data suggest that inhibition of GLAST expression in the spinal cord reduces excitatory synaptic activity and thereby spontaneous responses after nociceptive stimulation of the paw.

Ischemic preconditioning of free muscle flaps: an experimental study.

The aim of this study was to apply the hypothesis of ischemic preconditioning (IP) on free skeletal muscle (rat thigh flap). Five groups of Sprague-Dawley rats (n = 6) were used. In group A (control group), standard free autologous flap transfers were performed. Flaps in groups B and C underwent 4 and 6 h, respectively, of ischemia before transfer. In groups D and E, muscle flaps were preconditioned (3 x 10 min ischemia interrupted by 10 min of reperfusion, clip applied on the dissected artery of the flap) and subjected to 4 and 6 h, respectively, of ischemia before transfer. After 48 h of reperfusion, the muscle flaps were evaluated macroscopically as well as by histological and immunohystochemical staining. In group A, the viability was 100%, whereas in groups D and E the viability was 83.3% and 100%, respectively. Groups B and C had undergone macroscopically parceled to total necrosis, further confirmed by histological findings (fragmentation and disappearance of muscle striations, combined with tissue necrosis and intravascular thrombosis). The beneficial effect of IP demonstrated in the heart, liver, and small bowel extends to skeletal muscle, which can be used in free-flap transfers, if the transfer includes a long period of predictable ischemia.

Downregulation of cytosolic prostaglandin E2 synthase results in decreased nociceptive behavior in rats.

Nociception-evoked prostaglandin E2 (PGE2) release in the spinal cord contributes considerably to the development of hyperalgesia and allodynia. Biosynthesis of PGE2 involves the conversion of arachidonic acid to PGH2 by cyclooxygenases (COXs), followed by an isomerization of PGH2 to PGE2 by PGE2 synthases (PGESs). The roles of COX-1, COX-2, and the inducible microsomal PGES-1 have been studied in models of pain and inflammation. In contrast, in nociceptive processes, very little is known about the role of cytosolic PGES (cPGES), which has been described as being functionally coupled to COX-1. Here we show by in situ hybridization and immunohistological analysis that COX-1 and cPGES are constitutively expressed in neuronal and non-neuronal cells of the dorsal and ventral horns in the spinal cord of adult rats. The protein levels of both enzymes were not regulated by nociceptive stimuli; however, reduction of cPGES in rat spinal cord with intrathecal application of cPGES antisense oligonucleotides reduced the nociceptive behavior in zymosan-evoked thermal hyperalgesia and in the formalin assay. The data indicate that cPGES plays an important role in mediating early responses during spinal nociceptive processing.

Comparative proteomic analysis of the rat spinal cord in inflammatory and neuropathic pain models.

Pathological pain associated either with peripheral tissue damage and inflammation (inflammatory pain) or peripheral nerve injury (neuropathic pain) is characterized by persistent pain hypersensitivity. This hypersensitivity is believed to be mediated by sensitization of nociceptors and spinal dorsal horn neurons leading to hyperalgesia and allodynia. Changes of protein expression and/or phosphorylation are known to contribute to the development of this hyperexcitability of the nociceptive system. In the present study we analyzed protein patterns in the spinal cord following paw inflammation or sciatic nerve injury using two-dimensional (2D) gel electrophoresis combined with MALDI-TOF mass spectrometry. 2D-PAGE revealed nine and five regulated proteins following paw inflammation and sciatic nerve damage, respectively. These regulated proteins had not been identified previously with other methods. There was no overlap of regulated proteins between models except for the small heat shock protein alpha-crystallin, which was decreased in both models. In conclusion, this study illustrates that employment of the proteomic 2D-PAGE approach allows for identification of novel regulated proteins that may be involved in the central sensitization and possibly manifestation of chronic pain.