Kim Annink

36 Chapter 2 an experienced neuropathologist (M.L.). PCs were only counted when located in the Purkinje cell layer and when a clear nucleus was visible. PCs were categorized as: normal, abnormal or non-classified (Table 1 and Figure 2). The numbers of normal, abnormal and non-classified PCs were counted for each location using ImageJ software version 1.47 (Wayne Rasband, NIH, USA). Afterwards the numbers were corrected for the measured distance in cells per 100 µm. Also the percentage of abnormal PCs compared to normal PCs was calculated for every location. Table 1: Purkinje cell classification system. Category Definition Normal 1. An intact cell membrane 2. A basophilic/light stained cytoplasm 3. A clearly identifiable, basophilic/light stained nucleus with intact nuclear membrane 4. An identifiable nucleolus in the nucleus (In addition to 1 and 2, criteria 3 or 4 should be met to score a PC as normal) Abnormal 1. No intact/ruptured cell membrane 2. A hypereosinophilic stained cytoplasm 3. An identifiable nucleolus but no clear nucleus or rest of nucleolus Presence of activated Bergmann glia and shrinkage of total cell volume was taken into account as an additional confirmation of abnormality, but was not required Non-classified 1. The nucleolus and nucleus were not visible or could not be categorized 2. The soma of the PC was clearly visible 3. The cell is located in the PC layer