Copyright © 2005 American Medical Association. All rights reserved. From: Central Nervous System Injury–Induced Repulsive Guidance Molecule Expression in the Adult Human Brain Arch Neurol. 2005;62(10):1561-1568. doi:10.1001/archneur.62.10.1561 Figure Legend: Repulsive guidance molecule (RGM) antibodies. A, Domain of human RGM-A (GenBank accession numbers NP_064596 and NM_020211) detected by the RGM276-293 peptide-specific RGM antibody (gray-shaded letters). Numbers on the right indicate amino acids. B, Western blot analysis performed with RGM-A–transfected cell membranes. Unspecific staining with the secondary antibody was used for immunodetection (control). Incubation with anti-RGM–A antibody reveals the expected 43-kDa band (mouse RGM-A). Selected apparent molecular weights are indicated on the left. The arrow indicates the interface between the stacking and running gel. Date of download: 10/31/2017 Copyright © 2005 American Medical Association. All rights reserved.

Copyright © 2005 American Medical Association. All rights reserved. From: Central Nervous System Injury–Induced Repulsive Guidance Molecule Expression in the Adult Human Brain Arch Neurol. 2005;62(10):1561-1568. doi:10.1001/archneur.62.10.1561 Figure Legend: Repulsive guidance molecule (RGM) immunolocalization after central nervous system (CNS) injury. The specificity of the antibodies against RGM was confirmed by the inhibition of RGM immunoreactivity after preincubation for 3 hours on ice with the cognate peptide RGM276-293 (A) but not with the control peptide (B). C, In control brains, RGM immunoreactivity was detected on white matter–like fibers (similar to that obtained by myelin basic protein staining [data not shown]), oligodendrocytes, and the perikarya of some neurons. D, The RGM-immunopositive cells were also detected in the choroid plexus. E, Ependyma and frequently Purkinje cells of the cerebellum expressed RGM. After CNS injury of either ischemic or traumatic etiology, cellular RGM immunoreactivity revealed similar expression patterns. F, After CNS injury, RGM-immunopositive cells accumulated in infarctional white matter, hemorrhagic areas, the infarction core, and peri-infarctional areas. Arrow indicates infarction core. Early after ischemic damage (up to 2.5 days), RGM immunoreactivity was predominantly observed with (G) neurons and leukocytes of granulocytic, monocytic, and lymphocytic origin in vessels and infiltrative areas in ischemic tissue (H-J). Furthermore, in a widespread pattern, small and middle-sized vessels revealed RGM immunoreactivity confined to endothelial cells (H) and some fibromyocytes. I, Paralleled by edema formation, up to 1 to 7 days, RGM-immunopositive cells were found extravasating outside the vascular walls into the focal ischemic lesional parenchyma. Arrows indicate perpendicular invading routes. J, In perivascular regions, RGM-immunopositive cells formed clusters in Virchow-Robin–like spaces from days 1 to 7, which subsided later. K, At later stages, arising during the end of the first week after infarction, we detected RGM-immunopositive cells with fibroblast-like Morphologic features (arrow). L, Excessive RGM-immunopositive formations, constituting neo-laminae, were localized to areas of ongoing scar formation. M, With tissue reorganization of the lesion, “foamy,” lipid-laden, phagocytic RGM-immunopositive macrophages/lysosomal ingestions (arrow) were also observed. N, Double-labeling experiments identified RGM-immunopositive cellular structures localized to myelin basic protein–immunopositive myelin fibers and myelin basic protein–immunopositive oligodendrocytes. O, Most CD45+ leukocytes expressing RGM (70%) consist of CD68+ microglia/macrophages. With maturation of the lesion, we observed RGM co-expression by cellular and extracellular components of the developing scar. P, Abundant extracellular RGM-immunopositive laminae scar components were verified to be fibronectin immunopositive. Q and R, Oligodendroglial RGM expression was confirmed by additional 3′-cyclic nucleotide 3′-phosphodiesterase double labeling. Arrows indicate RGM-immunopositive cells and diaminobenzidine; arrowheads, 3′-cyclic nucleotide 3′-phosphodiesterase and fast blue (Q and R). Date of download: 10/31/2017 Copyright © 2005 American Medical Association. All rights reserved.

Copyright © 2005 American Medical Association. All rights reserved. From: Central Nervous System Injury–Induced Repulsive Guidance Molecule Expression in the Adult Human Brain Arch Neurol. 2005;62(10):1561-1568. doi:10.1001/archneur.62.10.1561 Figure Legend: Cumulative repulsive guidance molecule (RGM) expression in brains with focal cerebral ischemia (FCI). A, Regional analysis demonstrated significant accumulation of RGM-immunopositive cells in peri-infarctional border zones (P<.001), whereas in remote regions and in control brains, rare RGM-immunopositive cells were labeled. B, After FCI, quantitative assessment revealed an early, significant increase in RGM-immunopositive cell numbers during the first 24 hours followed by maximum RGM-immunopositive cell density from day 1.5 to day 2.5 after FCI. Subsequently, the numbers of lesional RGM-immunopositive cells, although decreasing, remained enhanced compared with controls for up to months after FCI. Control brains were used as the equivalent to 0 hours after FCI. The y-axis refers to total RGM-immunopositive cell counts (means) per high-power field. Error bars represent SEM. Date of download: 10/31/2017 Copyright © 2005 American Medical Association. All rights reserved.

Copyright © 2005 American Medical Association. All rights reserved. From: Central Nervous System Injury–Induced Repulsive Guidance Molecule Expression in the Adult Human Brain Arch Neurol. 2005;62(10):1561-1568. doi:10.1001/archneur.62.10.1561 Figure Legend: Mean cumulative repulsive guidance molecule (RGM) expression in brains with traumatic brain injury (TBI). A, Regional analysis demonstrated significant lesional accumulation of RGM-immunopositive cells in injured areas (P<.001), whereas in remote white matter regions and in control brains, only a few cells were labeled. B, The temporal RGM expression pattern demonstrated a cellular up-regulation already 1 hour after injury (P<.001), persisting at elevated levels for months after injury. Control brains were used as the equivalent to 0 hours after trauma. The y-axis refers to total RGM-immunopositive cell counts (means) per high-power field. Error bars represent SEM. Date of download: 10/31/2017 Copyright © 2005 American Medical Association. All rights reserved.