Details of the establishment and ongoing surveillance of the FHS' dementia-free cohort have been described previously [4
]. Participants found to be cognitively impaired are referred to the FHS' neurological group (RA, SS, SA, PAW) for annual neurological and NP examinations. If either the neurological or NP examination indicates the possibility of dementia, the case records are brought to a diagnostic review meeting. At this consensus conference, the diagnostic protocol requires review of all available medical information from 5 key sources: 1) FHS' health examinations, 2) FHS neurological examination, 3) NP assessments, 4) outside medical records and nursing home records and 5) for those whose brains come to autopsy, a post-mortem family interview. Post-mortem family interviews are generally conducted with the closest next of kin and include inquiries about changes in cognitive and functional status, and the time line associated with these changes. Further, questions from the Blessed Dementia Rating Scale [8
], the Hachinski ischemic score [9
] and the Retrospective Clinical Dementia Rating Scale [10
] are embedded in the Family Interview questionnaire. Presence or absence of dementia and the type of dementia are determined based on clinical information alone, and the clinical diagnosis is only shared with the neuropathologist at a clinico-pathological conference prior to which all pathological data and diagnoses have been independently recorded.
The severity of the cognitive impairment at death is categorized as mild cognitive impairment (MCI, usually corresponding to a CDR of 0.5) or dementia of varying severity (mild, moderate, severe corresponding roughly to CDR scores of 1, 2 and ≥3).
Diagnostic Criteria for Dementia, Alzheimer's Disease and MCI
All individuals identified as having dementia satisfy DSM IV criteria [11
]. Persons categorized as AD are required to meet NINCDS-ADRDA criteria for possible, probable or definite AD [12
]. The diagnosis of vascular dementia (VaD) is made based on ADDTC and NINDS-AIREN criteria, [13
] but the presence of vascular dementia does not disqualify a participant from obtaining a concomitant diagnosis of AD if indicated. Diagnostic criteria for other types of dementia such as Lewy Body disease and frontotemporal dementia are also carefully specified based on published criteria. [14
]. We have in the past defined MCI using a purely objective psychometrically-determined definition. However in this paper we use a standard definition of ‘probable’ MCI based on subjective and/or objective cognitive impairment in one or more cognitive domains with essentially normal functional status (so that the individual did not meet criteria for dementia). For each individual in the dementia surveillance system, a date of onset of cognitive impairment and a date of onset of dementia are assigned based on all available data as described above. Persons meeting criteria for MCI are also categorized as having either amnestic MCI with isolated memory or memory deficits along with other cognitive deficits, and non-amnestic MCI, with either a single or multiple non-memory deficits [16
Neuropathological evaluation of all autopsied brains is performed by a single neuropathologist (ACM) who is blinded to all demographic and clinical information. Briefly, the brains are received fresh and the gross neuropathological findings are recorded, including vascular lesions and the degree of atherosclerosis. The length of time between death and receiving the brain and/or placing it on ice varies widely based on whether the death occurred locally or in another state. The median postmortem delay was 6 hours, with the range being 1.5 to 72 hours and the interquartile range varying from 4 hrs (25%th ile) to 14.8 hours (75%th ile). The frontal, temporal and occipital poles are removed from one hemisphere and snap frozen at -80 C. The remaining tissue is fixed in 4% periodate-lysine-paraformaldehyde (PLP) at 4°C for at least 2 weeks. Ten micron paraffin-embedded sections from 30 brain regions, including: the olfactory bulb, 2 levels of the midbrain, 2 levels of pons, medulla, spinal cord, cervical spinal cord and 2 levels of the cerebellum; inferior frontal (Brodmann area (BA) 10,11,12), pre- and post-central (BA 4,3,2,1), inferior parietal (BA 39,40), anterior cingulate (BA 24), superior frontal (BA 9), dorsolateral middle frontal (BA 8), anterior temporal (BA 38), superior temporal (BA 20, 21,22), superior temporal posterior (BA 41,42), posterior cingulate (BA23,31), calcarine (BA 17,18), visual association (BA 19) and superior parietal (BA 7B) cortices; caudate, putamen, and nucleus accumbens (CAP), amygdala, entorhinal cortex (BA 28), the globus pallidus, substantia innominata, anterior hippocampus, hippocampus at the level of the lateral geniculate, and 2 levels of the thalamus are evaluated. Visual association area 19 is defined as the cortical region on the convexity, median and basal surfaces of the cerebrum directly caudal to the parieto-occipital fissure surrounding Brodmann visual area 18, also referred to as the peristriate area.
Histological Stains and Immunohistochemistry
Histological stains includes luxol fast blue, hematoxylin and eosin (LHE), Bielschowsky silver method, and immunocytochemistry for phosphorylated tau protein (Innogenetics, AT8, 1:2000), Aß protein (Dako, 6F-3D, 1:500, pretreated in 90% formic acid for 2 minutes), α- synuclein (Chemicon, affinity purified polyclonal, 1:3000, pretreated in formic acid) and αBcrystallin (Novocastra, NCL-ABCrys, 1:14,000).
Quantitation of Alzheimer's Disease Lesions
The density of NFT is rated semi-quantitatively in 14 regions using AT8 immunostained sections and in 13 sections using Bielschowsky stained sections. In the neocortical regions, i.e. inferior parietal (BA 40), middle frontal (BA 8), superior temporal (BA 22), calcarine (BA 17) and visual association (BA 19) cortices, a rating of 1+ corresponds to a maximum density of 1 NFT per 200 × field; 2+: 2-5 NFT/field; 3+: 6-9/field; and 4+: ≥10 NFT/field. For the medial temporal lobe structures, amygdala, entorhinal cortex and hippocampus, NFT are rated as follows: 1+: 1-10 NFT/field; 2+: 11-20 NFT/field; 3+: 21-30/field; 4+ ≥31/field. All determinations are made in areas of maximum involvement at a magnification of 200× using the average count from 3 microscopic fields. For NFT summary scores, the NFT density in the 4 or 5 neocortical areas plus the counts from the 3 medial temporal regions are tabulated. The density of NFT is also evaluated semi-quantitatively in the olfactory bulb, substantia innominata, substantia nigra, locus ceruleus, median raphe nuclei, and dorsal medullary nuclei using AT8 immunostained sections and the same density scale used for the neocortical regions.
The density of diffuse (DP) and neuritic (NPL) or compacted senile plaques is determined in the same regions. DP and NPL are rated separately as follows: a score of 1+ corresponds to a density of 1-9 plaques per 100 × microscopic field; 2+: 10-19 per field, 3+: 20-32 per field, and 4+: >32 plaques per field. All determinations are made by averaging the count in 3 microscopic fields in areas of maximum involvement at a magnification of 100×. The 1+ rating corresponds to a CERAD rating of sparse, a 2+ score corresponds to a CERAD rating of moderate, and a 3+ or 4+ score to a CERAD rating of frequent plaques [19
]. For a subset of participants, summary scores for DP and NPL in the 7 or 8 most affected regions, as well as for the neocortex and medial temporal lobe structures are also tabulated.
The distribution of NFT does not always strictly follow the hierarchical pattern described by Braak [20
]. Occasionally, modest NFT are found in the medial temporal lobe structures, while equal or greater densities of NFT are found in the neocortical regions. To compensate for non-hierarchical distributions of NFT, a quantitative NFT staging scheme was devised using the numerical sum of NFT density in the same brain regions outlined by Braak. The 6 levels of NFT severity, using AT8 immunostained sections, are defined as follows: Level I: summary score1-4; Level II: score 5-8; Level III: score 9-13; Level IV: score 14-18; Level V: score 19-23; Level VI: score 24-28.
The density of Lewy Bodies is evaluated using α-synuclein immunostained sections of the olfactory bulb, 2 levels of the substantia nigra, inferior parietal cortex, anterior cingulate cortex, middle frontal cortex, superior temporal cortex, amygdala, entorhinal cortex, transentorhinal cortex, substantia innominata, hippocampus, locus ceruleus, median raphe nuclei and dorsal medullary nuclei.
For the presence of argyrophilic grain disease, silver and AT8-positive medial temporal lobe (amygdala, entorhinal cortex and hippocampus) grains and αBcrystallin-positive ballooned neurons in the amygdala and entorhinal cortex are required.
Vascular and Microvascular Lesions
The FHS has been systematically documenting measures of vascular pathology and has developed a composite measure of vascular pathology derived using the National Alzheimer Co-ordinating Center (NACC) and University of California at Los Angeles (UCLA) ischemia score protocols and consistent with the Vascular Cognitive Impairment Harmonization guidelines [22
]. The developed ischemic injury scale (IIS) includes assessments of hippocampal sclerosis, volume of chronic infarcts, number of lacunes and microinfarcts, degree of atherosclerosis, arteriolosclerosis and white matter disease (including white matter integrity) and gives an overall single IIS score for each brain. Hippocampal sclerosis is judged by the presence of neuronal loss and gliosis in the hippocampal CA fields and subiculum using the following semi-quantitative scale: 0=none; 1=CA1 only; 2=CA1/Subiculum; 3=Subiculum fields/Prosubiculum; 4= All CA fields, subiculum/Prosubiculum. ‘Microinfarcts’ are defined as encephalomalacic lesions, 2 mm or smaller in greatest dimension, not identifiable on gross inspection of the brain. They are located in the cortex and subcortical white matter and include cavitated and non-cavitated chronic microinfarcts and microhemorrhages. Cavitated microinfarcts are defined as cystic areas of tissue loss or collapse with gliosis, and usually, macrophage infiltration. Non-cavitated microinfarcts are focal areas of cellular loss and gliosis without the formation of a cystic cavity. Microscopic deposits of blood or hemosiderin with minimal evidence of ischemic infarction are designated as microhemorrhages. The number of microinfarcts and microhemorrhages are tabulated in 5 neocortical regions and underlying white matter, hippocampus, entorhinal cortex, brainstem, and deep nuclei, including caudate, putamen, globus pallidus, thalamus, and amygdala. The number of microinfarcts and microhemorrhages are then recorded as a semiquantiative score per region: 0 = no microinfarcts; 1+ = 1-3 microinfarcts; 2+ = 4-8 microinfarcts; 3+ = 9-19 microinfarcts; 4+ = ≥20 microinfarcts. Degree of arteriolosclerosis is a composite score of the degree of hyaline thickening of arteriolar walls evaluated semi-quantitatively in 4 regions of deep white matter and basal ganglia. White matter disease is judged using a summary score of myelin loss and cribriform state evaluated in the subcortical white matter of 4 regions and the basal ganglia. Myelin loss is judged by gross inspection of the luxol fast blue, hematoxylin and eosin stained slide and rated semiquantitatively. If the area to be evaluated contains an infarct, the area is omitted from the analysis. Cribriform state is judged as a summary score of the following: 1. Perivascular rarefaction - the degree to which the tissue is attenuated or vacuolated around small blood vessels. 2. Dilation of perivascular spaces -widening of the perivascular space, using the greatest degree of dilation found around a single vessel seen in cross-section. 3. Perivascular macrophages- no perivascular macrophages = 0, 1-3 macrophages around a single small vessel = 1+, 4-8 macrophages = 2+, ≥ 9 macrophages = 3+. In addition to the measures that comprise the IIS, amyloid angiopathy is also evaluated in the leptomeninges and in the parenchyma in 4 neocortical regions: middle frontal, inferior parietal, superior temporal and calcarine cortices using a scale of severity modified from Von Sattel et al.
] and Esiri et al.