Tissue was sampled according to the location of a periventricular, subcortical, and NAWM ROI found across the 5 postmortem FLAIR slices. Five-µm-thick sections were stained with hematoxylin and eosin (H&E), Luxol fast blue-periodic acid Schiff (LFB-PAS), and by immunohistochemistry for myelin basic protein (MBP), phosphorylated neurofilament (pNF), glial fibrillary acidic protein (GFAP), microglia [ionized calcium-binding adaptor molecule (IBA1)], and collagen-IV. Antibody dilutions and manufacturer details are listed in Supplemental Methods m Supplemental Digital Content 3 , http://links.lww.com/NEN/A413 .
All slides were digitally scanned at 20x using the ScanScope XT (Aperio, Vista, CA.). ImageScope software (Aperio) was used to draw each ROI on the LFB-PAS slide that corresponded to the FLAIR ROI blind to the intensity level of the corresponding WMH. An area output was extracted from the original ROI and used to compare to the area of the corresponding postmortem FLAIR ROI. Each ROI from the LFB-PAS slide was copied and pasted to the same histologic position across all serially stained sections. Myelin, axonal and astroglial burden were analyzed using the Positive Pixel Count (PPC) algorithm designed to detect the brown hue and antibody saturation of 3,3’ diaminobenzidine (DAB) corresponding to the MBP, pNF, and GFAP, respectively (see Results for optimization of input parameters). The percentage positivities for myelin and axonal staining were normalized to the corpus callosum to control for any inter-subject staining variability. The resulting value was a percentage of positively stained pixels per area annotated. Vacuolation was also quantified using the PPC algorithm, but was modified to determine the percentage of space not occupied by neuropil, cells or vessels.
An advanced nuclear algorithm was modified to design 3 unique algorithms to count small vessels, activated microglia, and oligodendrocyte nuclei (Nuclear Algorithm 2004, Aperio). The algorithm used pixel count, roundness, elongation, and compactness to help detect the object of interest. The output contains a count, as well as area that were used to determine object density to remove ROI size as a confounding variable. Microglia were immunostained with IBA1 antibody (22 (link)) and operationally defined using morphologic characteristics and the optical density of the IBA1 stain (Nuclear v9, Aperio).
All slides were digitally scanned at 20x using the ScanScope XT (Aperio, Vista, CA.). ImageScope software (Aperio) was used to draw each ROI on the LFB-PAS slide that corresponded to the FLAIR ROI blind to the intensity level of the corresponding WMH. An area output was extracted from the original ROI and used to compare to the area of the corresponding postmortem FLAIR ROI. Each ROI from the LFB-PAS slide was copied and pasted to the same histologic position across all serially stained sections. Myelin, axonal and astroglial burden were analyzed using the Positive Pixel Count (PPC) algorithm designed to detect the brown hue and antibody saturation of 3,3’ diaminobenzidine (DAB) corresponding to the MBP, pNF, and GFAP, respectively (see Results for optimization of input parameters). The percentage positivities for myelin and axonal staining were normalized to the corpus callosum to control for any inter-subject staining variability. The resulting value was a percentage of positively stained pixels per area annotated. Vacuolation was also quantified using the PPC algorithm, but was modified to determine the percentage of space not occupied by neuropil, cells or vessels.
An advanced nuclear algorithm was modified to design 3 unique algorithms to count small vessels, activated microglia, and oligodendrocyte nuclei (Nuclear Algorithm 2004, Aperio). The algorithm used pixel count, roundness, elongation, and compactness to help detect the object of interest. The output contains a count, as well as area that were used to determine object density to remove ROI size as a confounding variable. Microglia were immunostained with IBA1 antibody (22 (link)) and operationally defined using morphologic characteristics and the optical density of the IBA1 stain (Nuclear v9, Aperio).
