Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation

Abstract

Demyelination in the adult CNS can be followed by extensive repair. However, in multiple sclerosis, the differentiation of oligodendrocyte lineage cells present in demyelinated lesions is often inhibited by unknown factors. In this study, we test whether myelin debris, a feature of demyelinated lesions and an in vitro inhibitor of oligodendrocyte precursor differentiation, affects remyelination efficiency. Focal demyelinating lesions were created in the adult rat brainstem, and the naturally generated myelin debris was augmented by the addition of purified myelin. After quantification of myelin basic protein mRNA expression from lesion material obtained by laser capture microdissection and supported by histological data, we found a significant impairment of remyelination, attributable to an arrest of the differentiation and not the recruitment of oligodendrocyte precursor cells. These data identify myelin as an inhibitor of remyelination as well as its well documented inhibition of axon regeneration.

Figure 1.

a, Areas of focal demyelination were created by stereotactic injection of 0.01% ethidium bromide into the CCPs of young adult Sprague Dawley rats (204–242 g). b, Three days later, myelin degeneration can be observed (toluidine blue-stained resin section). c, At this point, additional purified myelin, as confirmed by the presence of stacked membrane formations on electron micrographs of lipid droplets, was injected into the lesions. d, Fluorescently labeled myelin confirmed its ability to spread evenly throughout the entire lesion. For quantitative analysis of remyelination, 30-μm-thick cryostat sections were thaw mounted on PEP membrane slides and stained with 0.5% toluidine blue. e–g, The lesions on eight central sections were outlined (e), cut (f), and laser microdissected and captured (g) into a single Eppendorf tube. h, After extraction, the quality of the RNA was monitored by gel electrophoresis, and only samples of good quality were included in the subsequent analysis. Scale bar: (in h) a, 4 mm; b,50 μm; c, 0.72 nm; d, 600 μm; e–g, 377 μm.

Figure 2.

a, The extent of remyelination was quantified by qPCR for MBP relative toβ2-MG mRNA. There was no difference in relative MBP expression at day 14. However, at day 28, the relative MBP levels in myelin-treated animals failed to increase. Animals that received injections with equal amounts of purified liver membranes showed in termediate levels. b, Light microscopy on semithin toluidine blue-stained resin sections revealed successful remyelination. There are a small number of normally myelinated axons cut in transverse section and associated with the lesion edge at the bottom of this image; the vast majority of the remaining axons are surrounded by the thin dark blue-stained myelin sheaths typical of remyelination. c, In myelin-treated animals, remyelination was restricted to the lesion edge (bottom of image). The majority of the pale-staining axons remained demyelinated and surrounded by similarly pale-staining amorphous myelin debris (c, inset; h). d, e, In situ hybridization for PDGF-Rα showed comparable OPC densities (expressed as a percentage of cells in PBS controls) within lesions of myelin-treated animals and controls 8 d after lesion induction (d) but fewer cells expressing high levels of Nkx2.2 mRNA, a marker of OPC activation (e). f, The densities of SR-B+ macrophages was the same in myelin-treated and PBS control lesions. Electron microscopic analysis was used to establish the stage at which remyelination was arrested. g, Most axons in control lesions were reinvested with myelin sheaths formed by remyelinating oligodendrocytes. h, Although axons of myelin-treated animals were abundant and intact (albeit separated from one another by the presence of uncleared myelin debris), on electron micrographs they remained demyelinated. Processes (arrowheads; h) surrounding demyelinated axons (asterisk; i) were arrested at a premyelinatingstate and failed to produce compacted myelin. Scale bar: (in i) b, c, 50μm; g, 0.42μm; h, 2.8 μm; f, 0.28 μm. Error bars indicate SEM.