Three-dimensional stereological quantification of cell density and volume was determined essentially as described previously (Gemmell et al., 2012, 2014). We also ensured sections from the hippocampal formation from both CADASIL and controls were free of any direct tissue damage due to infarction. Thus, purely effects of remote ischemic injury were evaluated on the hippocampal formation. Briefly, 2 randomly selected 30-μm sections per sample were analyzed using a Visiopharm Integrator System (Visiopharm, Denmark). The precise regions of interest (ROI) in the EC (layer V, ECV) and hippocampus (cornu ammonis, CA1 and CA2; Fig. 1) were defined as previously described (Amaral and Insausti, 1990). Cell counts and volume estimations were conducted using a 100x oil-immersion objective on a Zeiss Axioplan Photomicroscope, whereas the XY and Z motorized stage (Prior ProScan II, Prior Scientific Instruments Ltd, UK) provided systematic random sampling. The unbiased dissector and nucleator method were used to estimate density and size of neurons and glial cells (Fig. 2). Tissue section thickness was measured using a Heidenhain gauge (Heidenhain GB Ltd, UK) at every 10th field during analysis. Approximately 40–80 fields per ROI were analyzed to achieve an identical sampling fraction for each ROI. Neurons were counted only when their nucleoli could be identified, and the cell body was either fully inside the dissector box measuring 56.43 × 45.14 μm or touching 1 of the 3 nonforbidden planes (right, top, and upper side) within the dissector height, which was set at 15 μm. SMI32-positive neurons were counted separately from the total neuron population.

The image shows how 3-dimensional stereology was used to quantify neuronal numbers and volumes. Neurons with nucleolus within counting frame or touching green lines were measured. The middle 15-μm depth was scanned through to identify neurons and glial cells within the dissector box. Glial cells and neurons with a nucleolus inside or touching the green lines were counted and measured for volume estimation. Neurons were marked with N, SMI32-positive neurons (colored brown) with S, and glial cells with G. The intersections of lines with cell membrane were marked by the researcher for volume estimation.

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