2.2 Neuropathologic assessments

All cases underwent neuropathologic assessment that included macroscopic and microscopic evaluation. Brains were sampled using a systematic and standardized protocol, in which neocortical samples were taken prior to brain dissection to assure uniformity of sampling and to obtain orthogonal sections of the cortical ribbon. Tissue sections were embedded in paraffin, and 5-μm thick sections were mounted on glass slides for histological examination and immunohistochemistry. Thioflavin-S fluorescent microscopy was used to assess AD-related pathology, which included counts of neurofibrillary tangles (NFT) and senile plaques in 6 cortical regions, 4 sectors of the hippocampus, 2 subregions of the amygdala and the basal nucleus of Meynert. Braak NFT stage [25] and Thal amyloid phase [26] were assigned based upon the distribution of NFT and senile plaques respectively, using previously published methods [27]. For diagnostic and staging purposes, immunohistochemistry was performed on all cases with an α-synuclein antibody (NACP, 1:3000 rabbit polyclonal, Mayo Clinic antibody) using a protocol (formic acid pretreatment and DAKO DAB polymer signal detection) that has been shown to be comparable, or better, than other methods [28]. Counts of Lewy bodies (at x200 magnification) were assessed in middle frontal, superior temporal, inferior parietal, cingulate and parahippocampal cortices, as well as the amygdala. Semi-quantitative ratings of the extent of the α-synuclein pathology and neuronal loss were made in the nucleus basalis of Meynert, substantia nigra and locus ceruleus..

When assigning subtypes of Lewy body disease, the presence, density, semi-quantitative scores and distribution of Lewy-related pathology followed recommendations of the Third Consortium for DLB criteria [19]. Transitional Lewy body disease (TLBD) included individuals with Lewy-related pathology in brainstem and predominantly limbic regions, and diffuse Lewy body disease (DLBD) included those with Lewy-related pathology in brainstem, limbic, and neocortical regions [19]. Patients were assigned a neuropathologic likelihood of DLB based on Lewy body disease subtype and severity of concurrent AD-type pathology according to Third Consortium for DLB criteria, as validated in a previous study [29].