Quantifying Myelin Thickness and Axon Myelination

Photomicrographs of sections through the corpus callosum were taken at 5800 × magnification. Eight to twelve images per animal were quantified. To measure the myelin thickness, we used Image Pro Premium software (Media Cybernetics, Rockville MD). A binary mask was first created using the minimum variance “auto dark” algorithm to select areas of positive myelin staining. Then, using the line profile function, two horizontal lines and two vertical lines were created and applied onto the same locations of each image mask. Only axons that fell on one of the four lines were evaluated. A total of 824 ± 136 axons were counted per animal. With the edge function, the rising length and falling length were recorded separately and sequentially. Here, we created a macro to standardize and automate the measurement of each image. The thickness of a myelin sheath was then calculated as T_myelin = L_falling− L_rising. The numbers myelinated axons and demyelinated axons were counted manually. Demyelinated axons were identified as having a diameter larger than 0.3 μm with no detectable compact myelin [71 (link), 103 (link)]. Axons smaller than 0.3 μm were excluded from analysis as potentially being normal unmyelinated fibers.