166 Chapter 6 recent project using the same tissue blocks [250, 251]. Four different stainings were used, each on one section of each tissue block: Bielschowsky Silver and SMI312 stainings to assess axonal density, orientation and integrity, and PLP and LFB to assess myelin integrity. Immunoreactivity was visualized by the avidin-biotin complex (ABC elite kit, PK6100; Vector Laboratories, California, United States) method using 3,3-diaminobenzidine (DAB; Sigma, Missouri, United States). Axonal density and orientation analysis Manual axon count and orientation analysis was done using the Bielschowsky silver staining in five of the ROIs: the reticular formation, the pyramidal tract, the proximal corpus callosum, the anterior cingulate gyrus, and the vermis. Within these regions the largest DTI differences have been reported [84, 104-107, 109, 238], and consistent fibre directions of the main axon bundles allowed for reliable axon counting. Methods for axon counting in the proximal corpus callosum, white matter of the anterior cingulate gyrus and vermis were adapted from Preziosa et al. [239]. Within these three ROIs, grids were drawn in QuPath with lines parallel and perpendicular to the main axon direction. Four circles (diameter 500 µm) were included centring around the intersections of the grid (Figure 2A). The grid was placed in a way that there were no large blood vessels or staining irregularities crossing the lines. Within these circles, the axons were counted that crossed the perpendicular and parallel lines (Figure 2B). An axon was only counted in one of the directions. If one axon crossed both lines, it was counted in the predominant direction (angle between axon and line >45 degrees). Besides absolute counts of axons we also calculated a ratio between the number of axons in the two directions. The axonal orientation ratio was used to investigate whether possible changes in axon counts were direction-specific. In the reticular formation and pyramidal tract ROIs, the direction of axons was in-plane to the cutting direction. The previously described method of counting axons passing the grid lines was not possible. Instead, we counted the axons in the outer quarter of each of the four circles (Figure 2C-D). The number of axons was calculated per 50000 µm2 of the ROI to account for possible area loss because of staining irregularities. Axon counting was performed while being blinded for the diagnosis of the donor (as in the other analyses). Additional colour intensity analyses were implemented on the Bielschowsky silver staining as a complementary measure to assess axonal integrity that was suitable for all ROIs of Table 1. For these analyses, we outlined an additional white matter ROI in each section, to calculate a 95% most colour intense
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