As previous imaging studies had suggested changes in the medial temporal lobe are associated with some memory deficits in patients with CADASIL, we concentrated on the hippocampal formation (Table 2). All the brain tissues were collected from the other centers and analyzed in Newcastle, preventing any sampling bias or introducing variation in the analysis. Temporal lobe blocks at the coronal level of the lateral geniculate nucleus were obtained for assessment of neuronal density. Cerebrovascular lesions including SVD pathology were assessed as described previously (Deramecourt et al., 2012). Sclerotic index was determined as described previously (Craggs et al., 2012). Axonal changes in the stem of the temporal lobe or temporal stem were assessed at the level of the anterior hippocampus in adjacent sections used to determine neuronal densities (Fig. 1). For neuronal counts and volumes, formalin-fixed, paraffin-embedded blocks were serially sectioned at thicknesses of 30 μm. They were immunostained using mouse anti–nonphosphorylated neurofilament H (anti-SMI32, Covance, CA; 1:500) monoclonal antibody to identify large pyramidal neurons and damaged axons in the white matter (WM) (Lindner et al., 2009; Voelker et al., 2004) and then counterstained with 0.1% cresyl fast violet to reveal the total neuron population.

Spectrum of hippocampal pathology in CADASIL casesa

Data show extent of hippocampal pathology (scores of 0–3; 0 = none, 1 = mild, 2 = moderate, 3 = severe).

Key: CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PVS, periventricular space.

(A) Diagram of the coronal block from one hemisphere of the cerebrum at the level of the anterior hippocampal formation; (B) Coronal fixed tissue slice from the cerebrum of a 79-year-old control participant at the level of the hippocampal formation taken for neuropathological examination; (C) Section stained with Nissl showing the complete hippocampal formation. (A, B) Boxes show delineated sample areas and (C) shows the anatomical regions of the temporal stem (TS), the entorhinal cortex layer V (ECV), and cornu ammonis (CA1 and CA2), which were assessed for pathological quantification. Abbreviations: DG, dentate gyrus; SB, subiculum.

To assess neurodegenerative pathology, sections were stained with hematoxylin and eosin for structural integrity and infarcts, Nissl and Luxol fast blue staining for cellular patterns and myelin loss, and Bielschowsky's silver impregnation and Gallyas stain for neuritic pathology. Amyloid-β and tau immunohistochemistry were performed to determine neuritic plaques and Braak staging of neurofibrillary tangles and rate ABC for amyloid β deposits (A), staging of neurofibrillary tangles (B), and scoring of neuritic plaques (C) scores as per National Institute on Aging–Alzheimer's Association guidelines (Table 2). Unless otherwise stated, all the tissues were processed for fixation in 10% buffered formalin and embedded in paraffin following standard protocols established in clinical histopathology services.

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