Acute- and late-phase matrix metalloproteinase (MMP)-9 activity is comparable in female and male rats after peripheral nerve injury.

BACKGROUND:In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. METHODS:Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. RESULTS:The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. CONCLUSION:The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes

The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury.

BackgroundMechanical 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.MethodsMass 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.ResultsFollowing 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.ConclusionsAfter 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

In Vivo Evaluation of Quantitative Percussion Diagnostics for Determining Implant Stability

PURPOSE: A percussion instrument (Periometer(®), Perimetrics LLC, Newport Beach, CA, USA) and rat model were used to test the hypothesis: percussion diagnostics provides reliable, reproducible indications of osseointegration. MATERIALS AND METHODS: Titanium implants were placed in femurs of 36 Sprague-Dawley rats. Each animal was assigned to one of six groups of six defined by one of three time points (2, 4, or 8 weeks post-placement) and one of two treatments (MMP inhibitor or vehicle). Percussion testing was conducted three times/subject at implant placement and at one of the time points. For each time point, there was an experimental group that received daily intraperitoneal injections of GM6001, and a control group that received no MMP inhibitor. The percussion data consisted of loss coefficient (LC) values that characterize energy dissipation. Statistical analysis was performed on the LC values for two animal groups using the paired Student t test to assess differences as a function of time, and the independent t test to compare mean LC for the study groups at sacrifice (α=0.05). Histological evaluation using the osteogenic CD40 protein marker was also performed. RESULTS: A nearly significant difference in mean LC at the 2-week time point was observed between the two treatments with the GM6001 group having the higher value (p = 0.053). There was a greater difference between the mean LC values for the 4-week GM6001 and vehicle groups (p = 0.001). The histological evidence for subjects in these two groups confirmed reduction of osteogenesis at the implant interface after administration of the MMP inhibitor. CONCLUSIONS: Lower vehicle LC values relative to the GM6001 therapeutic group were observed, consistent with the effect MMP inhibition has on matrix remodeling at the implant bone interface. This finding in conjunction with histological observations confirms that osseointegration can be monitored using percussion diagnostics

Cholesterol-Dependent LXR Transcription Factor Activity Represses Pronociceptive Effects of Estrogen in Sensory Neurons and Pain Induced by Myelin Basic Protein Fragments

BACKGROUND: A bioactive myelin basic protein (MBP) fragment, comprising MBP METHODS: In male and female normal and post-CCI rat sciatic nerves, we assessed: (i) cholesterol precursor and metabolite levels by lipidomics; (ii) MBP RESULTS: CCI regulated LXRα ligand and receptor levels in nerves of both sexes, with cholesterol precursors, desmosterol and 7-DHC, and oxysterol elevated in females relative to males. MBP CONCLUSION: The injury-released bioactive MBP fragments induce pronociceptive changes by selective inactivation of nuclear transcription factors, including LXRα. By Ncoa1 sequestration, bioactive MBP fragments render LXRα function to counteract pronociceptive activity of estrogen/ESR1 in sensory neurons. This effect of MBP fragments is prevalent in females due to high circulating estrogen levels in females relative to males. Restoring LXR activity presents a promising therapeutic strategy in management of neuropathic pain induced by bioactive MBP

A human coronavirus OC43‐derived polypeptide causes neuropathic pain

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV-OC43 p65-like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP-derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV-OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception-, immune-, and cellular energy-related activities. Affinity capture shows the association of OC43p with an Na+ /K+ -transporting ATPase, providing a potential direct target and mechanistic insight into virus-induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV-OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus-induced neuropathic pain

In vivo evaluation of quantitative percussion diagnostics for determining implant stability.

PurposeTo test in a rat model whether quantitative percussion diagnostics provide reliable, reproducible indications of osseointegration.Materials and methodsTitanium implants were placed in femurs of 36 Sprague-Dawley rats. Each animal was assigned to one of six groups defined by one of three time points (2, 4, or 8 weeks postplacement) and one of two treatments (matrix metalloproteinase [MMP] inhibitor GM6001 or control). Percussion testing was conducted three times per subject at implant placement and before sacrifice at one of the time points. For each time point, there was an experimental group that received daily intraperitoneal injections of GM6001, and a control group that received no MMP inhibitor. The percussion data consisted of loss coefficient (LC) values that characterize energy dissipation. Statistical analysis was performed on the LC values for the two animal groups using the paired Student t test to assess differences as a function of time, and the independent t test to compare mean LC for the study groups at sacrifice (α = .05). Histologic evaluation using the osteogenic CD40 protein marker was also performed.ResultsA nearly significant difference in mean LC at the 2-week time point was observed between the two treatments with the GM6001 group having the higher value (P = .053). There was a greater difference between the mean LC values for the 4-week GM6001 and control groups (P = .001). The histologic evidence for subjects in these two groups confirmed reduction of osteogenesis at the implant interface after administration of the MMP inhibitor.ConclusionsLower control LC values relative to the GM6001 therapeutic group were observed, consistent with the effect MMP inhibition has on matrix remodeling at the implant bone interface. This finding in conjunction with histologic observations confirms that osseointegration can be monitored using percussion diagnostics

Immunodominant fragments of myelin basic protein initiate T cell-dependent pain

AbstractBackgroundThe 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 resultsUsing 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.ConclusionsThese 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