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Background: Descending inhibitory pain control contributes to the endogenous defense against chronic pain and involves noradrenergic and serotonergic systems. The clinical efficacy of antidepressants suggests that serotonin may be particularly relevant for neuropathic pain conditions. Serotonergic signaling is regulated by synthesis, metabolisms, reuptake and receptors. To address the complexity, we used inbred mouse strains, C57BL/6J, 129 Sv, DBA/2J and Balb/c, which differ in brain serotonin levels. Results: Serotonin analysis after nerve injury revealed inter-strain differences in the adaptation of descending serotonergic fibers. Upregulation of spinal cord and midbrain serotonin was apparent only in 129 Sv mice and was associated with attenuated nerve injury evoked hyperalgesia and allodynia in this strain. The increase of dorsal horn serotonin was blocked by hemisectioning of descending fibers but not by rhizotomy of primary afferents indicating a midbrain source. Para-chlorophenylalanine-mediated serotonin depletion in spinal cord and midbrain intensified pain hypersensitivity in the nerve injury model. In contrast, chronic inflammation of the hindpaw did not evoke equivalent changes in serotonin levels in the spinal cord and midbrain and nociceptive thresholds dropped in a parallel manner in all strains. Conclusion: The results suggest that chronic nerve injury evoked hypernociception may be contributed by genetic differences of descending serotonergic inhibitory control.
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 PPAR gamma 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.
Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Cyclic GMP-dependent protein kinase 1 (PKG1) mediates presynaptic nociceptive long-term potentiation (LTP) in the spinal cord and contributes to inflammatory pain in rodents but the present study revealed opposite effects in the context of neuropathic pain. We used a set of loss-of-function models for in vivo and in vitro studies to address this controversy: peripheral neuron specific deletion (SNS-PKG1-/-), inducible deletion in subsets of neurons (SLICK-PKG1-/-) and redox-dead PKG1 mutants. In contrast to inflammatory pain, SNS-PKG1-/- mice developed stronger neuropathic hyperalgesia associated with an impairment of nerve regeneration, suggesting specific repair functions of PKG1. Although PKG1 accumulated at the site of injury, its activity was lost in the proximal nerve due to a reduction of oxidation-dependent dimerization, which was a consequence of mitochondrial damage in injured axons. In vitro, PKG1 deficiency or its redox-insensitivity resulted in enhanced outgrowth and reduction of growth cone collapse in response to redox signals, which presented as oxidative hotspots in growing cones. At the molecular level, PKG1 deficiency caused a depletion of phosphorylated cofilin, which is essential for growth cone collapse and guidance. Hence, redox-mediated guidance required PKG1 and consequently, its deficiency in vivo resulted in defective repair and enhanced neuropathic pain after nerve injury. PKG1-dependent repair functions will outweigh its signaling functions in spinal nociceptive LTP, so that inhibition of PKG1 is no option for neuropathic pain.
Persistent neuropathic pain is a frequent consequence of peripheral nerve injuries, particularly in the elderly. Using the IntelliCage we studied if sciatic nerve injury obstructed learning and memory in young and aged mice, each in wild type and progranulin deficient mice, which develop premature signs of brain aging. Both young and aged mice developed long-term nerve injury-evoked hyperalgesia and allodynia. In both genotypes, aged mice with neuropathic pain showed high error rates in place avoidance acquisition tasks. However, once learnt, these aged mice with neuropathic pain showed a significantly stronger maintenance of the aversive memory. Nerve injury did not affect place preference behavior in neither genotype, neither in young nor aged mice. However, nerve injury in progranulin deficient mice impaired the learning of spatial sequences of awarded places, particularly in the aged mice. This task required a discrimination of clockwise and anti-clockwise sequences. The chaining failure occurred only in progranulin deficient mice after nerve injury, but not in sham operated or wildtype mice, suggesting that progranulin was particularly important for compensatory adaptations after nerve injury. In contrast, all aged mice with neuropathic pain, irrespective of the genotype, had a long maintenance of aversive memory suggesting a negative alliance and possibly mutual aggravation of chronic neuropathic pain and aversive memory at old age.
