Kim Annink

55 Cerebellar injury in HIE: MRI versus histopathology photographed and analyzed using a standard light microscope (Zeiss AXIO Lab.A1, Oberkochen, Germany) and the ZEN 2.3 Lite software (Zeiss). H&E staining – scoring Purkinje cells Pictures were taken at 6 different locations in the vermis and cerebellar hemispheres: 1) at the apex and 2) the base of the anterior lobe, 3) at the apex and 4) the base of the posterior lobe and 5) at the apex and 6) the base of the flocculonodular lobe. All pictures were taken and analyzed at a magnification of 63 times. PCs were scored as normal or abnormal (Figure 2). A PC was scored normal when the nucleus was clearly visible, light stained with a clear nucleolus and the cellular cytoplasm was slightly eosinophilic and uniformly stained. A PC was scored abnormal when the nucleus was intensively dark stained and shrunken and when there was dark stained, hyper-eosinophilic cytoplasm. PCs were not included in the analyses when we were unable to identify a nucleus, because it was unclear whether this was due to hypoxic-ischemic injury or due to the way the slices were cut. In every picture the number of normal and abnormal PCs were counted, as well as the total number of PCs. The mean number and percentage for all the analyzed locations within the vermis and hemispheres were used for further analysis. PCs were counted manually using ImageJ (ImageJ program 1.47; National Institutes of Health, USA). In addition, the length of the PC layer was measured with ImageJ to correct for the length of the PC layer and number of PCs per 1000 µm. Figure 2: Examples of normal PCs (2A) and abnormal PCs (2B) in lobe 2 of the vermis of a patient with severe HIE who died five days after birth. 3