Cortical remyelination: a new target for repair therapies in multiple sclerosis

Abstract

Objective: Generation and differentiation of new oligodendrocytes in demyelinated white matter is the best described repair process in the adult human brain. However, remyelinating capacity falters with age in patients with multiple sclerosis (MS). Because demyelination of cerebral cortex is extensive in brains from MS patients, we investigated the capacity of cortical lesions to remyelinate and directly compared the extent of remyelination in lesions that involve cerebral cortex and adjacent subcortical white matter.

Methods: Postmortem brain tissue from 22 patients with MS (age 27-77 years) and 6 subjects without brain disease were analyzed. Regions of cerebral cortex with reduced myelin were examined for remyelination, oligodendrocyte progenitor cells, reactive astrocytes, and molecules that inhibit remyelination.

Results: New oligodendrocytes that were actively forming myelin sheaths were identified in 30 of 42 remyelinated subpial cortical lesions, including lesions from 3 patients in their 70s. Oligodendrocyte progenitor cells were not decreased in demyelinated or remyelinated cortices when compared to adjacent normal-appearing cortex or controls. In demyelinated lesions involving cortex and adjacent white matter, the cortex showed greater remyelination, more actively remyelinating oligodendrocytes, and fewer reactive astrocytes. Astrocytes in the white matter, but not in cortical portions of these lesions, significantly upregulate CD44, hyaluronan, and versican, molecules that form complexes that inhibit oligodendrocyte maturation and remyelination.

Interpretation: Endogenous remyelination of the cerebral cortex occurs in individuals with MS regardless of disease duration or chronological age of the patient. Cortical remyelination should be considered as a primary outcome measure in future clinical trials testing remyelination therapies.

Copyright © 2012 American Neurological Association.

Figures

Figure 1. Remyelination in Subpial Cortical Lesions

Brains from control patients without neurological disease have dense PLP immunoreactivity in all cerebral cortical layers (Panel A). Many regions of cortex in patients with MS show a similar pattern in PLP immunoreactivity (Panel B). A subpial demyelinated lesion with complete loss of PLP immunoreactivity is shown in Panel C. Many subpial lesions are not completely demyelinated, but contain PLP-positive myelin internodes (Panels D and E). Frequently, these areas contain actively-myelinating oligodendrocytes with PLP-positive cell bodies and processes extending to myelin internodes (Panels D and E, arrowheads in insets). Actively-myelinating oligodendrocytes are most frequent in lesions where PLP-positive myelin occupied 60% or less of the lesion area (Panel F). The scale bar represents 400 μm for Panels A-E.

Figure 2. Active Remyelination in Subpial Cortical Lesions

Subpial cortical lesions contain oligodendrocytes in various stages of differentiation (Panel A, arrows). Premyelinating oligodendrocytes with PLP-positive cell bodies and multiple processes are present (Panel B), but actively-myelinating oligodendrocytes with PLP-positive cell bodies and processes extending to multiple myelin internodes are more prevalent (Panels C and D). Myelin-internodal lengths are relatively short (Panels C and D), which supports the interpretation that these oligodendrocytes are actively remyelinating axons. Individual axons are ensheathed by multiple-short myelin internodes (Panel E, inset, arrows indicate nodes of Ranvier). Remyelinated fibers also show molecular maturation (Panel E, inset) as demonstrated by the appropriate distribution of the paranodal protein, Caspr (yellow in Panel E, inset; red in inset is PLP staining). The scale bar in Panel A represents 1 mm; the scale bars in Panels B - E represent 20 μm.

Figure 3. Remyelination in Chronic Leukocortical Lesions

PLP immunohistochemistry of a leukocortical lesion shows significant remyelination in the demyelinated cortex compared to the demyelinated white matter (Panel A, yellow line shows the boundary between cortex and white matter). MHC Class II staining (Panel B) identifies this lesion as chronic. The PLP-positive cells in demyelinated cortex have features of actively-remyelinating oligodendrocytes (Panel C is high magnification of the red box in Panel A), whereas those in demyelinated white matter often extend dystrophic processes with no apparent connection to myelin internodes (Panel D is high magnification of the blue box in Panel A). Twenty-eight chronic leukocortical lesions (49%) show evidence of remyelination (Panel E). Of these, 24 showed a greater extent of remyelination in the gray matter (GM) compared to that in the white matter (WM), and 17 of these 24 (70%) show evidence of active remyelination. The density of oligodendrocytes in the gray-matter portion of the actively-remyelinating lesions greatly exceeds the oligodendrocyte density in the white-matter portion (Panel F, P< 0.0001). The scale bars in Panels A and B represent 400 μm; the scale bars in Panels C and D represent 50 μm; the error bars in Panel F represent standard error of mean.

Figure 4. Comparison of the Astrocytosis and Inhibitors of Myelination in the Gray- and White-Matter Portions of Leukocortical Lesions

Gray-matter portions of leukocortical lesions have fewer reactive astrocytes compared to the white-matter portions. Panels A-C show the same lesion depicted in Figure 3. GFAP immunoreactivity is much greater in the white-matter portion of the lesion compared to the gray-matter portion (Panel A). The same pattern is seen in sections stained for CD44 (Panel B) and hyaluronan (Panel C). Occasional CD44-positive astrocytes were present in cortical lesions and frequently associate with blood vessels (Panel B, arrows). mRNAs encoding GFAP, CD44, and versican are significantly increased in white-matter portions of leukocortical lesions (WML) compared to normal-appearing white matter (NAWM) from the same patients with MS and white matter from controls without neurological diseases (CWM)(Panel D). Levels of these mRNAs are lower in the gray matter portions of leukocortical lesions (GML), normal appearing gray matter (NAGM), and control gray matter (CGM) (Panel D). Western blot analysis confirms increased protein levels of CD44 and versican in white-matter portions of leukocortical lesions (Panel E). The scale bar represents 500 μm for Panels A, B and C; the error bars in Panels D and E represent SEM; *= P<0.05, **= P<0.01 ***=P<0.005.

Figure 5. NG2-Postive Oligodendrocyte Progenitor Cells in Subpial Cortical Lesions

Oligodendrocyte progenitor cell density is not decreased in subpial-cortical lesions of MS. PLP immunoreactivity in cerebral cortex is shown from a control subject (Panel A), normal-appearing cortex from a patient with MS (Panel B), and a subpial lesion with no evidence of remyelination (Panel C). Panels D, E and F represent NG2 immunohistochemistry in sections cut adjacent to those in Panels A, B and C, respectively. NG2-positive oligodendrocyte progenitor cells are observed in all areas. Arrows indicate NG2-positive cell bodies; arrowheads indicate NG2-positive cells of blood vessels. The scale bars represents 50 μm.