Immunodominant myelin basic protein fragments induce mechanical hypersensitivity.
ABSTRACT: An insulating myelin sheath ensures saltatory conduction of mechanosensory A afferents. Myelin damage results in the electrical instability of A fibers and the ability to generate pain in response to light touch/pressure (mechanical allodynia). We have hypothesized and then established that the release of T cell epitopes of myelin basic protein (MBP) enables nociceptive circuitry in myelinated fibers. Thus, mass spectrometry analysis of the rat sciatic nerve proteome followed by bioinformatics examination of the datasets revealed a loss of MBP and activation of T-helper cell signaling in the nerves undergoing chronic constriction injury (CCI). Matrix metalloproteinase-9 (MMP-9) proteolysis resulted in the MBP digest peptides, including the MBP84-104 and MBP68-86 regions, which exhibit prominent immunogenic epitopes. Myelin-forming Schwann cells and paranodal areas accumulated MHCII, MMP-9 and the degraded MBP at the sciatic nerve injury site. Administration of the immunodominant MBP84-104 and MBP68-86 peptides but not of the control peptides in a naïve rat sciatic nerve produced robust mechanical allodynia. Allodynia was accompanied by the T cell infiltration and an increase in MHCII, IL-17A and TNF- levels at the nerve injection site and the segmental ganglia. The pro-nociceptive activity of the synthetic MBP84-104 diminished in athymic nude rats lacking T cells. SB-3CT, an antagonist of MMP-9, inhibited mechanical allodynia, neuroinflammation and spinal sensitization after CCI. Collectively, our novel data implicate, for the first time, MMP-mediated cleavage of MBP and the resulting MBP digest fragments as a major cause of neuropathic pain. Overall design: Gene extression profiling of total RNAs extracted from rat sciatic nerves, dorsal root ganglion and spinal cords after MBP84-104 peptide injection
INSTRUMENT(S): Agilent-028279 SurePrint G3 Rat GE 8x60K Microarray (Probe Name version)
Project description:An insulating myelin sheath ensures saltatory conduction of mechanosensory A afferents. Myelin damage results in the electrical instability of A fibers and the ability to generate pain in response to light touch/pressure (mechanical allodynia). We have hypothesized and then established that the release of T cell epitopes of myelin basic protein (MBP) enables nociceptive circuitry in myelinated fibers. Thus, mass spectrometry analysis of the rat sciatic nerve proteome followed by bioinformatics examination of the datasets revealed a loss of MBP and activation of T-helper cell signaling in the nerves undergoing chronic constriction injury (CCI). Matrix metalloproteinase-9 (MMP-9) proteolysis resulted in the MBP digest peptides, including the MBP84-104 and MBP68-86 regions, which exhibit prominent immunogenic epitopes. Myelin-forming Schwann cells and paranodal areas accumulated MHCII, MMP-9 and the degraded MBP at the sciatic nerve injury site. Administration of the immunodominant MBP84-104 and MBP68-86 peptides but not of the control peptides in a naïve rat sciatic nerve produced robust mechanical allodynia. Allodynia was accompanied by the T cell infiltration and an increase in MHCII, IL-17A and TNF- levels at the nerve injection site and the segmental ganglia. The pro-nociceptive activity of the synthetic MBP84-104 diminished in athymic nude rats lacking T cells. SB-3CT, an antagonist of MMP-9, inhibited mechanical allodynia, neuroinflammation and spinal sensitization after CCI. Collectively, our novel data implicate, for the first time, MMP-mediated cleavage of MBP and the resulting MBP digest fragments as a major cause of neuropathic pain. Gene extression profiling of total RNAs extracted from rat sciatic nerves, dorsal root ganglion and spinal cords after MBP84-104 peptide injection
Project description:BACKGROUND:The myelin sheath provides electrical insulation of mechanosensory Aβ-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aβ-fibers is believed to activate the nociceptive circuitry in Aβ-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood. METHODS AND RESULTS:Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. CONCLUSIONS:These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.
Project description:Myelin basic protein (MBP) is an auto-antigen able to induce intractable pain from innocuous mechanical stimulation (mechanical allodynia). The mechanisms provoking this algesic MBP activity remain obscure. Our present study demonstrates that membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) releases the algesic MBP peptides from the damaged myelin, which then reciprocally enhance the expression of MT1-MMP in nerve to sustain a state of allodynia. Specifically, MT1-MMP expression and activity in rat sciatic nerve gradually increased starting at day 3 after chronic constriction injury (CCI). Inhibition of the MT1-MMP activity by intraneural injection of the function-blocking human DX2400 monoclonal antibody at day 3 post-CCI reduced mechanical allodynia and neuropathological signs of Wallerian degeneration, including axon demyelination, degeneration, edema and formation of myelin ovoids. Consistent with its role in allodynia, the MT1-MMP proteolysis of MBP generated the MBP69-86-containing epitope sequences in vitro. In agreement, the DX2400 therapy reduced the release of the MBP69-86 epitope in CCI nerve. Finally, intraneural injection of the algesic MBP69-86 and control MBP2-18 peptides differentially induced MT1-MMP and MMP-2 expression in the nerve. With these data we offer a novel, self-sustaining mechanism of persistent allodynia via the positive feedback loop between MT1-MMP and the algesic MBP peptides. Accordingly, short-term inhibition of MT1-MMP activity presents a feasible pharmacological approach to intervene in this molecular circuit and the development of neuropathic pain.
Project description:Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting saltatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e., mechanical allodynia). Our earlier (Liu et al., 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84-104) into an intact sciatic nerve produces a robust and long-lasting (>30days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel α2δ1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system.
Project description:Sciatic nerve chronic constriction injury (CCI) in rodents produces nerve demyelination via proteolysis of myelin basic protein (MBP), the major component of myelin sheath. Proteolysis releases the cryptic MBP epitope, a demyelination marker, which is hidden in the native MBP fold. It has never been established if the proteolytic release of this cryptic MBP autoantigen stimulates the post-injury increase in the respective circulating autoantibodies. To measure these autoantibodies, we developed the ELISA that employed the cryptic 84-104 MBP sequence (MBP84-104) as bait. This allowed us, for the first time, to quantify the circulating anti-MBP84-104 autoantibodies in rat serum post-CCI. The circulating IgM (but not IgG) autoantibodies were detectable as soon as day 7 post-CCI. The IgM autoantibody level continually increased between days 7 and 28 post-injury. Using the rat serum samples, we established that the ELISA intra-assay (precision) and inter-assay (repeatability) variability parameters were 2.87% and 4.58%, respectively. We also demonstrated the ELISA specificity by recording the autoantibodies to the liberated MBP84-104 epitope alone, but not to intact MBP in which the 84-104 region is hidden. Because the 84-104 sequence is conserved among mammals, we tested if the ELISA was applicable to detect demyelination and quantify the respective autoantibodies in humans. Our limited pilot study that involved 16 female multiple sclerosis and fibromyalgia syndrome patients demonstrated that the ELISA was efficient in measuring both the circulating IgG- and IgM-type autoantibodies in patients exhibiting demyelination. We believe that the ELISA measurements of the circulating autoantibodies against the pathogenic MBP84-104 peptide may facilitate the identification of demyelination in both experimental and clinical settings. In clinic, these measurements may assist neurologists to recognize patients with painful neuropathy and demyelinating diseases, and as a result, to personalize their treatment regimens.
Project description:: In this study, the phytochemical composition and the possible prophylactic effects of an aqueous ethanol extract of Haematoxylon campechianum flowers (HCF) on peripheral neuropathic pain in a chronic constriction injury (CCI) rat model are investigated. Rats with induced CCI were subjected to neuropathic pain behaviour tests and evaluated by chemical, thermal, and mechanical sensation tests and functional recovery of the brain stem and sciatic nerve at 7- and 14-day intervals. The effect of the extract on acute pain and inflammation is also investigated. The extract exerted both peripheral and central analgesic and anti-inflammatory properties in addition to antipyretic effects that are clear from targeting COX, LOX and PGE. It was found that CCI produced significant thermal and mechanical hyperalgesia, cold allodynia and deleterious structural changes in both sciatic nerve and brain stem. Treatments with HCF extract significantly improved cold and thermal withdrawal latency, mechanical sensibility and ameliorated deleterious changes of sciatic nerve and brain stem at different dose levels. The extract also ameliorated oxidative stress and inflammatory markers in brain stem and sciatic nerve. It suppressed the apoptotic marker, p53, and restored myelin sheath integrity. The effects of HCF extract were more potent than pregabalin. Fifteen secondary metabolites, mainly gallotannins and flavonoids, were characterized in the extract based on their retention times and MS/MS data. The identified phenolic constituents from the extract could be promising candidates to treat neuropathic pain due to their diverse biological activities, including antioxidant, anti-inflammatory and neuroprotective properties.
Project description:Mechanical pain hypersensitivity associated with physical trauma to peripheral nerve depends on T-helper (Th) cells expressing the algesic cytokine, interleukin (IL)-17A. Fibronectin (FN) isoform alternatively spliced within the IIICS region encoding the 25-residue-long connecting segment 1 (CS1) regulates T cell recruitment to the sites of inflammation. Herein, we analyzed the role of CS1-containing FN (FN-CS1) in IL-17A expression and pain after peripheral nerve damage.Mass spectrometry, immunoblotting, and FN-CS1-specific immunofluorescence analyses were employed to examine FN expression after chronic constriction injury (CCI) in rat sciatic nerves. The acute intra-sciatic nerve injection of the synthetic CS1 peptide (a competitive inhibitor of the FN-CS1/α4 integrin binding) was used to elucidate the functional significance of FN-CS1 in mechanical and thermal pain hypersensitivity and IL-17A expression (by quantitative Taqman RT-PCR) after CCI. The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves.Following CCI, FN expression in sciatic nerve increased with the dominant FN-CS1 deposition in endothelial cells, Schwann cells, and macrophages. Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve. CS1 peptide inhibited the LPS- or starvation-stimulated activation of the stress ERK/MAPK pathway in cultured Schwann cells.After physical trauma to the peripheral nerve, FN-CS1 contributes to mechanical pain hypersensitivity by increasing the number of IL-17A-expressing (presumably, Th17) cells. CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.
Project description:Neuropathic pain genesis is related to gene alterations in the dorsal root ganglion (DRG) after peripheral nerve injury. Transcription factors control gene expression. In this study, we investigated whether octamer transcription factor 1 (OCT1), a transcription factor, contributed to neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. Chronic constriction injury produced a time-dependent increase in the level of OCT1 protein in the ipsilateral L4/5 DRG, but not in the spinal cord. Blocking this increase through microinjection of OCT1 siRNA into the ipsilateral L4/5 DRG attenuated the initiation and maintenance of CCI-induced mechanical allodynia, heat hyperalgesia, and cold allodynia and improved morphine analgesia after CCI, without affecting basal responses to acute mechanical, heat, and cold stimuli as well as locomotor functions. Mimicking this increase through microinjection of recombinant adeno-associated virus 5 harboring full-length OCT1 into the unilateral L4/5 DRG led to marked mechanical allodynia, heat hyperalgesia, and cold allodynia in naive rats. Mechanistically, OCT1 participated in CCI-induced increases in Dnmt3a mRNA and its protein and DNMT3a-mediated decreases in Oprm1 and Kcna2 mRNAs and their proteins in the injured DRG. These findings indicate that OCT1 may participate in neuropathic pain at least in part by transcriptionally activating Dnmt3a and subsequently epigenetic silencing of Oprm1 and Kcan2 in the DRG. OCT1 may serve as a potential target for therapeutic treatments against neuropathic pain.
Project description:Neuropathic pain occurs after peripheral nerve damage, inflammation or infection. In this situation, microglial cells become activated and play a key role in producing pain. Minocycline (microglia inhibitor), was reported to reduce pain when used preventively. However, it seems that, when used after nerve injury, results in its pain reducing effects are different. In this regard, to assess the pain reducing differences of minocycline, neuropathic pain was induced by the ligation of the sciatic nerve in the rat which is recognized as chronic constriction injury (CCI) and minocycline was administered before and after sciatic nerve injury. Wistar male rats (200-250 g, n=6) were used in these experiments. Rats were distributed in various groups: vehicle-treated CCI (control), sham-operated and minocycline-treated CCI groups. In the first part of the experiment (pre-injury study), minocycline (10, 20, 30 and 40 mg/kg,) was injected one hour before surgery and then daily for two weeks. In the second part (post injury study), minocycline was administered: 1: at day one after nerve damage once a day to day 14, 2: at day seven after surgery and continued daily until day 14. Analgesimeter for thermal hyperalgesia and von Frey hairs for mechanical allodynia were used to evaluate pain behavior. Thermal hyperalgesia and mechanical allodynia were attenuated significantly, when minocycline used before surgery, while it was not able to reduce pain behavior administered after surgery. It seems that, in spite of what some previous studies have reported, here, minocycline is not able to attenuate established neuropathic pain.
Project description:Purpose:Neuropathic pain involves injury or alteration of the normal sensory and modulatory nervous systems to produce a set of symptoms that are often difficult to treat. Previous study indicates that crocin has anti-inflammatory properties that may be mediated by the neurotransmitter system. In this study, we determine if there is an interaction between crocin and the cannabinoid system on ?chronic ?constriction ?injury (CCI)?-induced neuropathic pain in male rats. Materials and Methods:In this experimental study, adult male Wistar rats (220-250 g) were used. CCI was induced by setting four loose ligatures around the ?sciatic nerve. In part 1, after nerve lesion, vehicle, ?crocin (60 mg/kg) or Win 55-212-2 (0.1 mg/kg) as an agonist and AM 251 (0.1 mg/kg) as an antagonist of cannabinoid receptors were injected intraperitoneally daily in separate groups for 2 weeks. In part 2, two weeks after nerve lesion, vehicle (5 µL), crocin (6 µg/5 µL), Win 55-212-2 (0.1 µg/5 µL), AM 251 (0.1 µg/5 µL) were administered intracerebroventricularly (ICV) in separate groups. Mechanical allodynia and thermal hyperalgesia were measured using Von Frey filaments and plantar test device, respectively, at day 14. Data were analyzed by ?two-way ANOVA and Sidak's multiple comparisons post-test. Results:Results indicated that centrally administered crocin significantly decreased thermal hyperalgesia and mechanical allodynia. Also, peripheral injection of ?crocin significantly decreased mechanical allodynia but not thermal hyperalgesia. Central or peripheral administration of Win 55-212-2 or AM 251 modulates the analgesic effect of ?crocin significantly. Conclusion:Our findings showed that ?crocin ?has significant analgesic effects that are probably mediated by an endocannabinoid mechanism.