Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: C3d+ microglial clusters in chronic MS cases with slowly expanding lesions. Slowly expanding white matter lesion in a chronic MS case (chronic MS#1), showing (A) loss of Luxol fast blue (LFB) staining (asterisk), (B) loss of immunoreactivity for the myelin oligodendrocyte glycoprotein (MOG, asterisk) (C) with intracellular inclusions of MOG+ myelin degradation products inside ionized calcium‐binding adapter molecule 1 (IBA‐1)+ macrophages (arrows) at the lesion edge and (D) loss of immunoreactivity for the proteolipid protein (PLP, asterisk). Lesion edge is indicated by the line in A,B,D. (E) Perivascular “cuffing” with CD3+ T‐lymphocytes, expanding within the Virchow‐Robin space of capillaries, located within the lesion or at the perilesional area. (F) Human leukocyte antigen (HLA‐DR)+ microglia accumulated at the lesion edge (indicated by the line), whereas the lesion core was generally hypocellular (asterisk). (G,H) Consecutive sections stained for (G) C3/C3b/iC3b/C3d and (H) CD45, showing corresponding profiles of (G) C3d+ linear deposits (arrows) and (H) linear clusters of microglia (arrows). (I) Double immunolabeling for C3/C3b/iC3b/C3d (green) and CD45 (red) showing that a cluster of CD45+ microglia surrounds the C3d+ linear deposit. Nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). (J) Double immunolabeling for C3/C3b/iC3b/C3d (red) and PLP (green) showing that C3d is deposited on axons which are partially immunoreactive for PLP, indicative of partial demyelination. (K) Confocal microscopy of double immunolabeling for CD45 (red) and PLP (green), demonstrating localization of PLP within a CD45+ microglial cluster. (L) Confocal microscopy of triple immunolabeling for C3/C3b/iC3b/C3d (red), CD45 (blue) and PLP (green), showing C3d deposition on a partially demyelinated axon within a CD45+ microglial cluster. (M,N) C3d deposits (green) on amyloid precursor protein (APP)+ axons or terminal bulbs (red), indicative of transection or impaired protein transport. Nuclei are stained with DAPI (blue). Arrows in I‐N point to sites of markers colocalization. Scale bars: (A,B,D,F) 200 μm; (E,G,H) 100 μm; (C,J–L) 50 μm; (I,N) 20 μm; (M) 10 μm. Hematoxylin was used as counterstain in B,D–H. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: Staining, Binding Assay, Immunolabeling, Confocal Microscopy

Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: Acute MS cases lack C3d+ microglial clusters. Actively demyelinating lesions in an acute MS case (acute MS#2), showing (A) loss of Luxol fast blue (LFB, asterisk) staining, and (B) accumulation of CD68+ macrophages in the lesion core (asterisk). Lesion edge is indicated by the line in A,B. (C) Immunoreactivity for the myelin oligodendrocyte glycoprotein (MOG) showed a general loss of staining within the lesion (asterisk), (D) with MOG+ myelin degradation products (arrows) at the lesion edge and in the lesion core. (E) Immunostaining for proteolipid protein (PLP), showing a patchy pattern (asterisk), indicative of active demyelination. (F–I) T‐lymphocyte immunostaining, showing (F) “cuffing” of CD3+ T‐lymphocytes around blood vessels and (G) abundance in the parenchyma. (H) Most of these T‐lymphocytes were CD8+ whereas (I) CD4+ staining, although present, was less pronounced and detected only at the perivascular spaces. (J) Myelin‐associated glycoprotein (MAG)+ myelin products were found in the lesion core (arrows) where (K) CD68+ cells had phagocytic morphology (arrows), and (L) at the lesion edge (arrows) where (M) we observed CD68+ cells of morphology consistent with activated microglia (black arrow) and phagocytic macrophages (white arrows). (N) Periplaque area with normal appearing white matter (NAWM), showing sparse ionized calcium‐binding adapter molecule 1 (IBA‐1)+ microglia with nonreactive/nonphagocytic morphology, (O) confirmed by the small morphology of CD68 reactive cells in the same area. C3/C3b/iC3b/C3d immunostaining, showing (P) abundant immunoreactivity within macrophages in the lesion core, (Q) in a staining pattern consistent with debris. Scale bars: (A,B) 200 μm; (C,E,G,N,P) 50 μm; (H,I,K,M,O) 20 μm; (F,J,L,Q) 10 μm; (D) 5 μm. Hematoxylin was used as counterstain in C–Q. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: Staining, Immunostaining, Binding Assay

Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: C3d+ microglial clusters are associated with neuronal C3 production in chronic MS cases. (A) A subset of neurons in a chronic MS case with slowly expanding white matter lesions, showing C3/C3b/iC3b/C3d immunoreactivity in a punctate pattern consistent with the neuronal secretory machinery (arrows), suggesting neuronal synthesis of C3. (B) In situ hybridization for C3 mRNA (red) and immunostaining for neuronal nuclei (NeuN, blue) proving production of C3 mRNA by neurons. (C) Double immunolabeling showing colocalization of C3/C3b/iC3b/C3d (red) and the endoplasmic reticulum marker Calnexin (green), further supporting neuronal synthesis of C3. (D) Double immunolabeling of C3/C3b/iC3b/C3d (green) and amyloid precursor protein (APP, red) showing neuronal localization. Nuclei in C and D are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). Scale bars: (A,B) 10 μm; (C,D) 5 μm. Hematoxylin was used as counterstain in A. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: In Situ Hybridization, Immunostaining, Immunolabeling, Marker, Staining

Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: C3d+ microglial clusters and neuronal C3 in ischemic stroke cases with advanced lesions. (A–D) Ischemic stroke case with initial white matter lesions showing (A) loss of Luxol fast blue (LFB) staining from the lesion core (asterisk), (B) preservation of immunoreactivity for proteolipid protein (PLP, asterisk) and (C) preservation of immunoreactivity for pan‐neurofilament (NF, asterisk). (D) Microglial cells, expressing the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox marker of oxidative stress, were accumulated at the lesion edge forming an ill‐defined rim in initial lesions whereas the lesion core was generally hypocellular (asterisk). (E) Advanced white matter stroke lesions showed massive accumulation of p22phox+ macrophages at the lesion edge (white asterisk) and some microglia/macrophages within the lesion (black asterisk). Lesion edge is indicated by the line in A,D,E. (F) In ischemic stroke cases with initial lesions, C3d was deposited on axons located close to blood vessels exclusively. (G,H) In ischemic stroke cases with advanced lesions, we found (G) ionized calcium‐binding adapter molecule 1 (IBA‐1)+ microglial clusters in areas corresponding to (H) linear C3d deposits. (I) Double immunolabeling for C3/C3b/iC3b/C3d (green) and human leukocyte antigen (HLA‐DR, red) showed that HLA‐DR+ microglial clusters surround the C3d+ linear deposits (arrows point to sites of markers co‐localization). Nuclei are stained with 4',6‐diamidino‐2‐phenylindole (DAPI, blue). (J) In advanced stroke lesions, punctate staining for C3/C3b/iC3b/C3d was detected at the neuronal bodies (arrows) in a subset of neurons located in close proximity to a lesion. Scale bars: (A,D,E) 200 μm; (B,C) 100 μm; (G–I) 50 μm; (F) 20 μm; (J) 10 μm. Hematoxylin was used as counterstain in A‐C,F–H,J. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: Staining, Preserving, Expressing, Marker, Binding Assay, Immunolabeling

Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: C3d+ microglial clusters and neuronal C3 in the mouse brain at 1 week after TBI. Sagittal sections of mouse brain at 1 week after experimental closed head injury at a distance from the impact site. (A) Loss of Luxol fast blue (LFB) staining (asterisks) and (B) LFB+ myelin degradation products (arrows), indicative of demyelination. (C) Immunostaining for proteolipid protein (PLP) showing reactivity. (D) Hematoxylin and eosin (H&E) staining showing no signs of hemorrhage. (E,F) Immunostaining for the pan‐neurofilament (NF) marker of axons, showing a punctate staining pattern (white arrows in F) or disrupted axonal profiles (black arrows in F), consistent with axonal transection. (G,H) Immunostaining for ionized calcium‐binding adapter molecule 1 (IBA‐1) showing microglial clusters (arrows). (I–K) Immunoreactivity for (I) C3/C3b/iC3b/C3d (red) was detected within (J) IBA‐1+ microglial clusters (green) as indicated by the sites of markers co‐localization (arrows in K). Nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). (L) In situ hybridization for C3 mRNA (red) and immunostaining for neuronal nuclei (NeuN, blue) showing localization of the C3 mRNA signal in the cytoplasm of NeuN+ cells, indicating local synthesis of C3 by neurons. Scale bars: (A,C,D,E,G) 100 μm; (F,H,I–K) 20 μm; (B,L) 10 μm. Hematoxylin was used as counterstain in C–H. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: Staining, Immunostaining, Marker, Binding Assay, In Situ Hybridization

Journal: Glia

Article Title: Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

doi: 10.1002/glia.23090

Figure Lengend Snippet: Primary Antibodies, Dilution, Source

Article Snippet: C3/C3b/iC3b/C3d , 2/11 , 1:100b † , Hycult Biotech.

Techniques: