Ultrastructural Analysis of Remyelinated Corpus Callosum

For electron microscopic examination, Epon embedding was performed as previously described [23 (link)]. In brief, the rats (n = 4 for each group) were transcardially perfused with 2% glutaraldehyde (Gla) and 2.5% PFA in 0.1 mol/l PBS. Brains were quickly removed and placed on ice. The corpus callosum was dissected and placed in 3% Gla in 0.1 M cacodylate buffer (pH 7.4) at 4 °C overnight and transferred to 1% osmium tetroxide in the same buffer for 1 h at room temperature. Tissue was transversely cut into 1-mm blocks that were fixed in osmium tetroxide at 4 °C overnight, dehydrated through ascending ethanol washes, and embedded in epoxy resin. To study the remyelinated axons of the corpus callosum, serial 1-μm semi-thin sections were cut and stained with 1% toluidine blue and examined by light microscopy. To analyze myelin sheaths in the corpus callosum, 60~70-nm-thick ultra-thin sections were stained with uranyl acetate and lead citrate prior to examination by tEM (FEI Tecnai G2 T12, USA), and the image was analyzed by TEM Imaging and Analysis. For morphometric analysis, the axonal diameter (d) as the shortest distance across the center of the axon was measured. The axonal diameter plus the total myelin sheath thickness on both sides was defined as the fiber diameter (D). The g-ratio was calculated using the d/D ratio.