168 Chapter 6 Figure 2. Axon counting analysis methods. Axon counting was performed in the proximal corpus callosum, anterior cingulate gyrus as displayed in panels A-B, and in the reticular formation and pyramidal tract as displayed in panels C-D. Axons were counted four times for each individual in the same regions of interest (ROIs) that were also used for the colour intensity and are percentage stained analyses. (A) Exemplary grid for the axon counting analyses in the proximal corpus callosum. Axons were counted when crossing the straight red lines. (B) Zoomed version of A. Each yellow circle corresponds to a counted axon in the main fibre direction crossing the grid. The absolute axon count in the analyses was defined as the sum of yellow axonal crossings. Angulated axons are counted in green and served as the denominator for calculating the axonal orientation ratio. The axonal orientation ratio was used to investigate whether possible changed axon counts were direction-specific. (C) Exemplary grid for the axon counting analyses in the reticular formation. (D) Zoomed version of C with in-plane axons highlighted in yellow. The scale bar represents 100 µm in A and C and 50 µm in B and D.
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