Elevated activity and microglial expression of myeloperoxidase in demyelinated cerebral cortex in multiple sclerosis

Abstract

Recent studies have revealed extensive cortical demyelination in patients with progressive multiple sclerosis (MS). Demyelination in gray matter lesions is associated with activation of microglia. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we examined the extent of microglial activation in the cerebral cortex and the relationship of microglial activation and MPO activity to cortical demyelination. Twenty-one cases of neuropathologically confirmed multiple sclerosis, with 34 cortical lesions, were used to assess microglial activation. HLA-DR immunolabeling of activated microglia was significantly higher in demyelinated MS cortex than control cortex and, within the MS cohort, was significantly greater within cortical lesions than in matched non-demyelinated areas of cortex. In homogenates of MS cortex, cortical demyelination was associated with significantly elevated MPO activity. Immunohistochemistry revealed MPO in CD68-positive microglia within cortical plaques, particularly toward the edge of the plaques, but not in microglia in adjacent non-demyelinated cortex. Cortical demyelination in MS is associated with increased activity of MPO, which is expressed by a CD68-positive subset of activated microglia, suggesting that microglial production of reactive oxygen species is likely to be involved in cortical demyelination.

Figure 1

Cortical demyelination in multiple sclerosis, demonstrated by labeling of paraffin sections for myelin basic protein. A,B. Multiple well‐defined areas of subpial and transcortical demyelination (arrows) in sections of temporal (A) and frontal (B) cortex. C. Subpial plaque. The arrows indicate the pial surface. D. Intracortical plaque. E. Leucocortical plaque. The arrows indicate the junction between the cerebral cortex and underlying white matter. F. Transcortical plaque. The arrows indicatie the junction between cortex and white matter.

Figure 2

Distribution of HLA‐DR‐positive cells in cerebral cortex. A. This section of cortex from a control brain includes only a single HLA‐DR‐positive cell (arrow), adjacent to a capillary. B. Non‐demyelinated cortex from a patient with multiple sclerosis includes sparsely distributed HLA‐DR‐positive microglia (arrows). C. Multiple activated microglia are labeled in a region of cortical demyelination.

Figure 3

Comparison of HLA‐DR‐ and CD68‐labeling in cerebral cortex. A. CD68‐positive cells are present within a cortical plaque (top left part of figure) but not in the adjacent, non‐demyelinated cortex. The labeled cells are most numerous along the edge of the plaque. B. At higher magnification, CD68 is seen to be present in rounded cell bodies with little labeling of finer cell processes. C. In a serial section through the edge of the plaque illustrated in A, antibody to HLA‐DR labels more cells than that are positive for CD68 both within the central part of the plaque and along its edge. The labeling extends along the cytoplasmic processes of many of the cells. HLA‐DR‐positive cells are also present, albeit at lower density, in the adjacent, non‐demyelinated cortex.

Figure 4

HLA‐DR‐labeling of plaques, non‐demyelinated multiple sclerosis (MS) cortex and control cortex. The mean values ± SE are shown for 32 demyelinated and 14 non‐demyelinated samples of MS cortex and 20 control samples. The labeling is significantly greater within plaques than that in matched non‐demyelinated areas of cortex in the same sections (P = 0.0016). HLA‐DR immunolabeling is higher in all regions of MS than control cortex but the difference is statistically significant only for the comparison of demyelinated with control cortex (P < 0.05). Abbreviation: gm = grey matter.

Figure 5

Levels of active myeloperoxidase in relation to extent of demyelination in frozen tissue blocks. The mean values ± SE are shown for nine blocks with mild, nine with moderate and 14 with severe demyelination. There is no significant difference between the groups.

Figure 6

Levels of active myeloperoxidase in control, non‐demyelinated and demyelinated multiple sclerosis (MS) cortex. The mean values ± SE are shown for 20 control samples, 14 of non‐demyelinated and 32 of demyelinated MS cortex. Myeloperoxidase levels are significantly associated with cortical demyelination (P = 0.0008) but not with MS in the absence of demyelination.

Figure 7

Immunofluorescent labeling (green) of myeloperoxidase (MPO) (A), CD68 (D) and HLA‐DR (E) in a cortical plaque with dye 4′6′‐diamidino‐2‐phenylindole nuclear counterstain (blue in B, E, F). The merged images are shown in C, F and I. The MPO labeling is cytoplasmic and granular, in keeping with the lysosomal location of this enzyme and appears similar in distribution to that for CD68. HLA‐DR labeling is more abundant than either MPO or CD68.

Figure 8

Double immunofluorescent labeling of myelin basic protein (MBP) (green in A,E), neurofilament (NF) (green in I), glial fibrillary acidic protein (GFAP) (green in M), CD68 (green in Q) and myeloperoxidase (MPO) (red in B, F, J, N,R) in a cortical plaques, with dye 4′6′‐diamidino‐2‐phenylindole (DAPI) nuclear counterstain (blue in C, G, K, O, S). D, H, L, P and T are merged images. Examination of A–D shows MPO‐positive cells to be concentrated near the edge of a cortical plaque, demarcated by the labeling of MBP in adjacent non‐demyelinated cortex. At higher magnification (E–H), MBP is clearly visible within some of the MPO‐positive cells, which appear yellow in the merged image. The MPO‐positive cells are separate from the neurofilament‐positive neuronal somata and processes in the cortical plaque illustrated in I–M. MPO‐positive cells are also distinct from the GFAP‐positive astrocytes in the plaque shown in M–P. In contrast, most of the MPO‐positive cells near the edge of a cortical plaque co‐localize with CD68 (Q–T).