Figure 3.

Effects of fingolimod on remyelination following lysolecithin-induced demyelination. A: Representative images of demyelinated slices, control or treated with fingolimod (100 pmol/L) for 14 DIV postlysolecithin, immunostained against myelin (MBP; red) and axons (NFM; green). Fingolimod increases the amount of myelin associated with axons compared with control. Scale bar = 20 μmol/L. B: Quantification of amount of myelin expressed as area of MBP immunostaining (μm²) per ×63 objective image normalized to control culture values at 14 DIV postlysolecithin. Fingolimod treatment (100 pmol/L, 1 μmol/L) for 14 DIV postlysolecithin significantly increases the amount of myelin compared with control cultures. *P < 0.05. C: Electron micrograph of demyelinated slice treated with 100 pmol/L fingolimod for 14 DIV postdemyelination indicates thin myelin sheaths (arrows) suggestive of remyelination. Thin myelin sheaths at the paranode (PR) adjacent to the node of Ranvier (NR) are suggestive functional remyelination. Scale bar =1 μm. D: Electron micrograph of demyelinated slice treated with 100 pmol/L fingolimod for 14 DIV postdemyelination indicates maintenance of appropriate cytoarchitecture, with synaptic terminals (ST) onto an axon (Ax) complete with synaptic vesicles, with an adjacent astrocyte (As) likely mediating homeostatic functions at the synapse. Scale bar = 0.5 μm. E: Toluidine-blue stained image of demyelinated slice allowed to recover for 14DIV postdemyelination. Arrowheads indicate demyelinated axons. Arrows indicate thin myelin sheaths suggestive of remyelination. Scale bar = 5 μm. MD, myelin debris F: Toluidine-blue stained image of demyelinated slice treated with 100 pmol/L fingolimod for 14 DIV postdemyelination demonstrates a healthy neuron (N) and oligodendrocyte (O), as well as many thin myelin sheaths (arrows) indicating remyelination. Scale bar = 5 μm.