Figure 5

Pro-nociceptive activity of MBP peptides. (A) The 1–171 sequence of human MBP (GenBank #AAH08749). The immunogenic regions are shown at the bottom of the panel using human MBP residue numbering. Following MBP cleavage by MMP-9, the mass and, consecutively, the sequence of the digest fragments was determined by MALDI-TOF MS. The italicized numbers indicate the positions of the cleavage sites. (B) von Frey testing after the intraneural injection of MBP peptides (50 μg in 5 μL) or vehicle (PBS) into a naïve rat sciatic nerve. Within 1 day a decline in mechanical withdrawal thresholds was observed after the MBP84-104 and MBP68-86 injection. Control MBP2-18 and MBP84-104 scrambled (sMBP84-104) peptide induced no significant change in thresholds compared to PBS. The mean withdrawal thresholds (gram force; g) ± SEM of n = 6/group (**, P < 0.01, *; P < 0.05). (C) Methylene Blue Azure II staining in 1-μm-thick sciatic nerve sections. MBP84-104 produced myelin splitting and cell infiltration 3 days post-injection into the naïve nerve. Uncompromised axons, surrounded by a compact rim of myelin are observed in the nerves after the sMBP84-104 injection. Representative micrographs of n = 4/group. Scale bars, 20 μm. (D) Immunostaining of MHCII (green) or T cell receptor (TCR, green) in the nerve at 3 days after the MBP injection into the intact nerve. Scale bars, 25 μm. The graph represents morphometry of the mean MHCII-positive cell numbers in the sciatic nerves ± SEM of n = 4/group and three sections per n (*, P < 0.05). (E) Immunostaining of MHCII (green) and Iba1 in the monocytes/macrophages (red) or S100 in the Schwann cells (red) in the nerve after the MBP84-104 injection in the wild-type rats. Macrophages (arrowheads) and Schwann cells (arrows) represent MHCII-reactive cells in the nerve, exposed to the immunodominant MBP84-104 peptide. DAPI, blue. Representative micrographs of n = 4/group. Scale bars, 10 μm.