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A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways.
The stimulation of the AMP-activated kinase (AMPK) by 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) has been associated with antihyperalgesia and the inhibition of nociceptive signaling in the spinal cord in models of paw inflammation. The attenuated nociception comes along with a strongly reduced paw edema, indicating that peripheral antiinflammatory mechanisms contribute to antinociception. In this study, we investigated the impact of AICAR on the immune cell composition in inflamed paws, as well as the regulation of inflammatory and resolving markers in macrophages. By using fluorescence-activated cell sorting (FACS) analysis and immunofluorescence, we found a significantly increased fraction of proresolving M2 macrophages and anti-inflammatory interleukin (IL)-10 in inflamed tissue, while M1 macrophages and proinflammatory cytokines such as IL-1 were decreased by AICAR in wild type mice. In AMPKα2 knock-out mice, the M2 polarization of macrophages in the paw was missing. The results were supported by experiments in primary macrophage cultures which also showed a shift to a proresolving phenotype with decreased levels of proinflammatory mediators and increased levels of antiinflammatory mediators. However, in the cell cultures, we did not observe differences between the AMPKα2+/+ and −/− cells, thus indicating that the AICAR-induced effects are at least partially AMPK-independent. In summary, our results indicate that AICAR has potent antiinflammatory and proresolving properties in inflammation which are contributing to a reduction of inflammatory edema and antinociception.
Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.
The long-chain fatty acid receptor FFAR1 is highly expressed in pancreatic β-cells. Synthetic FFAR1 agonists can be used as antidiabetic drugs to promote glucose-stimulated insulin secretion (GSIS). However, the physiological role of FFAR1 in β-cells remains poorly understood. Here we show that 20-HETE activates FFAR1 and promotes GSIS via FFAR1 with higher potency and efficacy than dietary fatty acids such as palmitic, linoleic, and α-linolenic acid. Murine and human β-cells produce 20-HETE, and the ω-hydroxylase-mediated formation and release of 20-HETE is strongly stimulated by glucose. Pharmacological inhibition of 20-HETE formation and blockade of FFAR1 in islets inhibits GSIS. In islets from type-2 diabetic humans and mice, glucose-stimulated 20-HETE formation and 20-HETE-dependent stimulation of GSIS are strongly reduced. We show that 20-HETE is an FFAR1 agonist, which functions as an autocrine positive feed-forward regulator of GSIS, and that a reduced glucose-induced 20-HETE formation contributes to inefficient GSIS in type-2 diabetes.
The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18:1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18:1, 9-and 13-HODE and HETEs.
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.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
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.
Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages.
Background
Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation.
Results
In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice.
Conclusion
Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.