Kim Annink

56 Chapter 3 H&E staining – scoring DN The DN pictures were taken at three different locations spread throughout the structure, also with a magnification of 63 times. Neurons in the H&E stained slices of the DN were also scored normal or abnormal using the same criteria as for PC scoring (see above). Only cells with a transcellular diameter of at least 14 µm were taken into account, because cell characteristics were not reliably assessable in smaller cells. The DN cell counts (normal, abnormal and total) were corrected for the measured surface. H&E staining – cytotoxic edema The H&E stained slices of the cerebellar hemispheres, vermis and DN were visually scored for cytotoxic edema. Cytotoxic edema was scored as absent (no, 0 points), minimal or focal cytotoxic edema (mild, 1 point), diffuse cytotoxic edema (moderate, 2 points) or very extensive cytotoxic edema (severe, 3 points) by an experienced neonatal pathologist. HLA-DR and CD68 staining The slices stained with monoclonal antibodies for HLA-DR and CD68 were scored according to a previously published method (9). The slices of the cerebellar hemispheres, vermis and DN were scored based on the magnification that was necessary to visualize the stained microglia/macrophages: 0 points were given if there was no staining visible (no staining), 1 point when staining was visible with a magnification of 200x (mild staining), 2 points in case a magnification of 100x was sufficient to visualize staining (moderate staining) and 3 points if the staining was visible with a magnification of 25x (severe staining). Statistical analysis Statistical analysis was performed using IBM SPSS Statistics for Windows, version 25 (IBM Armonk, New York, USA). The correlation between the ADC measurements of the two raters was calculated using the Spearman’s Rho test. Mean ADC values between infants with HIE and controls, were compared using the independent t-test for parametric data and Mann-Whitney-U test for non-parametric data. The histopathological measurements (number of normal PCs per 1000µm and percentage of normal PCs of the total PCs) were compared between the different locations in the hemispheres and vermis using Repeated Measures ANOVA (between