Figure 1.
Loss of AQP4 and Cx43 in perivascular lesions of neuromyelitis optica (NMO) in case NMO-4
(A–M) Immunoreactivities for AQP4 and GFAP are markedly diminished around blood vessels in the ventral side of the pons (A, B). In contrast, myelin is preserved around the same blood vessels in the perivascular areas as assessed by KB staining (C). Complement components are specifically deposited in the affected perivascular spaces (D, insert). Serial sections of affected blood vessel from the same case (E–M). Perivascular inflammatory infiltrations contain a large number of neutrophils, mononuclear cells (inset to E), CD68-positive macrophages (E, F) and few eosinophils. Myelin proteins, including Cx47 (G), MOG (H), OSP (I) and MAG (J), are preserved around the vessels. AQP4 and Cx43 are completely absent in highly degenerative, GFAP-positive astrocytes (K–M). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 0.4 mm (A–D); 100 µm (E–M).
Figure 2.
Loss of Cx47 and Cx32 in chronic active NMO lesions in case NMO-6
(A–M) Low magnification view of KB staining (A) and immunostainings for MOG (B), Cx47 (C) and Cx43 (D) in the cerebrum. Immunoreactivity for Cx47 is extensively diminished in the demyelinating lesion with cavitation in the cerebral white matter, whereas immunoreactivity for MOG is relatively preserved (A–C). This lesion is classified as pattern D for Cx43 and pattern A for Cx47/Cx32. Up-regulation of Cx43 immunoreactivity is observed in the corresponding demyelinating area (D). Arrows indicates cavitation and arrowheads show the lesion boundary (C, D). CD68-positive foamy macrophages are observed at the periphery and perivascular areas of the lesion. No foamy macrophages contain KB-positive granules within their cytoplasm (E, F, insert). Immunoreactivities for MAG and OSP are decreased in these lesions (G, H). Expression levels of Cx47 and Cx32 are extensively reduced in the lesion (I, J) compared with the non-affected white matter (K, L). Numerous Cx43- positive astrocytes are present in this lesion suggesting astrogliosis. Cx43 immunoreactivity is also preserved in the perivascular areas (M). Scale Bar = 2 mm (A–D); 20 µm (E); 100 µm (F–J, M); 50 µm (K, L).
Figure 3.
Coexistence of distal oligodendrogliopathy in active NMO lesions in case NMO-4
(A–L) CD68 immunostaining demonstrates massive infiltration of macrophages in the cerebral peduncle (A, arrows). Immunoreactivity for MOG is relatively preserved but is completely lost for MAG (B, C, arrows). Higher magnification of the lesion (D–L). Immunoreactivity for MOG is relatively preserved in contrast to complete loss of MAG in this lesion (D, E). Cx47 expression is diminished compared with non-affected white matter (F). Lesion boundary areas (G–I). A dotted line indicates the boundary. Immunostainings for Cx47 and Cx32 are slightly diminished but preserved inside the lesion (G, H). Immunostaining of Nogo-A is markedly decreased and apoptotic nuclear condensation of oligodendrocytes is present (I, insert). Immunoreactivities for AQP4 and Cx43 within highly degenerative GFAP-positive astrocytes are completely lost (J–L). This lesion is classified as pattern B for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm(A-C); 0.5 mm (D–F); 50 µm (G, H); 100 µm (I); 200 µm (J–L).
Figure 4.
Distal oligodendrogliopathy and astrocytopathy in anti-AQP4 antibody-seropositive NMO (case NMO-10).
In active lesions of the cerebral white matter, KB staining and MOG immunostaining show remaining myelin (A, B). Patterns of preferential MAG loss and marked loss of GFAP immunoreactivity are seen (C, D). Higher magnification reveals sharply demarcated, prominent MAG loss in this lesion with infiltration of numerous CD68-positive macrophages, whereas immunoreactivity for MOG, MBP and OSP is preserved in the lesion (E–I). Immunoreactivity for Cx47 is diminished compared with non-affected white matter (J). Complete loss of AQP4 and Cx43 in degenerative, GFAP-positive astrocytes (K–M) and complement deposition are observed around blood vessels with perivascular cell cuffing (N, O). Complement components are present within foamy macrophages in this lesion (P). Nogo-A-positive oligodendrocytes are markedly decreased in this lesion and some remaining oligodendrocytes show nuclear condensation, suggesting apoptotic changes (Q). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm (A–D); 200 µm (E–M); 50 µm (N, O); 20 µm (P, Q).
Figure 5.
Cx43 and AQP4 astrocytopathy in active lesions of MS (case MS-3).
Low magnification view of GFAP (A) and Cx43 (B) immunostaining in the pons. Immunoreactivity for Cx43 is markedly diminished in multiple lesions covered by GFAP-positive astrocytes (A, B, arrows). Massive perivascular cuffing, mostly consisting of lymphocytes, is observed in these lesions (C). Immunoreactivities for Cx32 and Cx47 are decreased in these lesions (D, E) compared with non-affected white matter (D, E, insert). Loss of MAG compared with MOG and OSP is prominent (F–H). Infiltration of macrophages phagocytosing myelin debris, which are immunopositive for myelin proteins (F, H, insert). Numerous GFAP-positive hypertrophic astrocytes exist in perivascular areas and parenchyma of these lesions (I). Patchy loss of AQP4 and diffuse loss of Cx43 in the center and periphery of lesions (J, K). Some hypertrophic astrocytes demonstrate membranous staining for AQP4 (J, insert). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Scale Bar = 4 mm (A, B); 50 µm (C); 100 µm (D–K).
Figure 6.
Loss of oligodendrocytic Cx47 and Cx32 expression in chronic lesions of MS (case MS-5).
KB staining in a chronic inactive lesion of the optic nerve (A). Higher magnification view of the lesion boundary area (B–I, corresponding to Figure 5A, square). Immunoreactivities for Cx47 and Cx32 are diminished beyond the demyelinated area (B, C) as revealed by immunostaining for MOG, OSP and MAG (D–F). This lesion is classified as pattern D for Cx43 and pattern A for Cx47/Cx32. In contrast, MLC1, AQP4 and Cx43 expression are up-regulated because of astrogliosis (G–I). MLC1 is localized at perivascular foot processes even in chronic gliotic tissues (G). Scale Bar = 1 mm(A); 200 µm (B–I).
Figure 7.
Neuropathological findings in a case with Marburg’s variant of MS (case MS-6).
Huge, active demyelinating lesions with perivascular lymphocytic cuffing (A) and infiltrating foamy macrophages phagocytosing myelin debris (B, corresponding to Figure 6A, square). Expression of Cx43 is extensively lost in this lesion (C, corresponding to Figure 6A, square). (D–I) Higher magnification view of the square area in Figure 6C. Loss of MAG is more obvious than for other myelin proteins including MBP and MOG (D–F). Numerous GFAP-positive hypertrophic astrocytes covering active lesions show patchy loss of AQP4 expression and diffuse loss of Cx43 expression (G–I). This lesion is classified as pattern A for Cx43 and pattern B for Cx47/Cx32. Nogo-A-positive oligodendrocytes are decreased and demonstrate apoptotic nuclear condensation (J, insert). Immunoreactivity for phosphorylated neurofilaments reveals degenerated, transected axons with axonal spheroids in the lesion (K). Immunoreactivity for MLC1 is specifically confirmed in perivascular foot process termini in non-affected white matter (L). This staining pattern is no longer observed in the lesion center and only cell bodies of gemistocytic astrocytes are immunopositive for MLC1 (M). Creutzfeldt astrocytes forming multiple micronuclei show a loss of Cx43 despite the preservation of GFAP and AQP4 (N–Q). Double immunofluorescence staining for Cx43 and Cx47 in an active lesion (R, S). In the unaffected white matter, Cx43 and Cx47 expression is well preserved in the perivascular space and white matter (R). Double staining for Cx43 and Cx47 shows partial juxtaposition or colocalization suggestive of GJ plaque formation around oligodendrocytes (inset). In contrast, immunoreactivity for Cx43 is markedly diminished in the perivascular space of inflamed blood vessels while immunoreactivity for Cx47 is preserved (S). Asterisks indicate vascular lumen (R, S). Scale Bar = 1 mm(A); 500 µm (B, C); 200 µm (D–I); 50 µm (J, K); 100 µm (L, M); 10 µm (N–Q); 20 µm (R); 50 µm (S).
Figure 8.
Relationship of inflammatory components with patterns of Cx43 loss in MS and NMO lesions.
(A, B) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (A) or chronic active (B) lesions of MS cases according to Cx43 patterns. (C, D) Positivity rates of perivascular lymphocytic cuffing or perivascular deposition of complement and immunoglobulin in active (C) or chronic active (D) lesions of NMO/NMOSD cases according to Cx43 patterns. Patterns N lesions are excluded in this figure.