Antemortem MRI findings correlate with hippocampal neuropathology in typical aging and dementia

Recruitment and characterization of subjects.

Subjects studied were participants in the Mayo Rochester Alzheimer’s Disease Research Center (ADRC) and Mayo Alzheimer’s Disease Patient Registry (ADPR).11 These are ongoing longitudinal studies of aging and dementia that provide a formal mechanism for identification, enrollment, and longitudinal clinical characterization of both community and referral subjects who are cognitively intact and subjects who are impaired. Both the ADRC and ADPR protocols include MRI studies, and autopsy permission is sought as well. Studies were performed with Mayo institutional review board approval and informed consent of the subject or an appropriate surrogate.

The 67 subjects studied represent an unselected series of individuals participating in the ADRC/ADPR who had undergone both an antemortem MRI examination and also had an autopsy. Assessment of mental status examination performance was assessed with the Mini-Mental State Examination (MMSE). All participants were categorized by clinical diagnosis during consensus committee meetings consisting of a geriatrician (E.G.T.), neurologists (R.C.P., B.F.B., E.K.), neuropsychologists (G.E.S.), psychometrists, and research nurses who had seen the patient. Although clinical diagnoses of specific dementing disorders were assigned, for purposes of this study we simply categorized each subject’s antemortem clinical state as cognitively normal, demented, or mild cognitive impairment (MCI).12,13⇓

Patients with a suspected cognitive impairment were identified during general medical examinations by Mayo primary care physicians. A neurologist (R.C.P., B.F.B., E.K.) then performed a detailed neurologic examination and obtained a complete history from the patient and a collateral source. Neuropsychological tests were administered to assess memory, attention, language, visuospatial skills, and problem solving. Patients were not excluded for the presence of ongoing medical problems such as diabetes, hypertension, or heart disease. The diagnosis of dementia was made according to the Diagnostic and Statistical Manual, 3rd. ed., revised (DSM-III-R) criteria14 at the consensus conference.

The diagnosis of MCI was a clinical judgment based on the following criteria: 1) memory complaint documented by the patient and collateral source; 2) normal general cognition as determined by measures of general intellectual function and mental status screening instruments; 3) normal activities of daily living; 4) not demented (according to the DSM-III-R)14; 5) memory impairment15; and 6) Clinical Dementia Rating score of 0.5.12,13,16⇓⇓

Cognitively normal subjects were recruited from the same pool of patients coming to Mayo primary care physicians for a general medical examination. Normal subjects were evaluated in the same way as cognitively impaired patients. Their status was reviewed at the consensus conference. Subjects categorized as cognitively normal had no active neurologic or psychiatric disorders. Like the patients, some had ongoing medical problems; however, the illnesses or their treatments did not interfere with cognitive function.

MRI methods.

All subjects were imaged at 1.5 Tesla (Signa, General Electric Medical Systems, Milwaukee, WI) using a standardized imaging protocol. Measurements of intracranial volume were derived from a T1-weighted sagittal sequence with 5-mm contiguous sections. Volume measurements of the hippocampi were derived from a T1-weighted, three-dimensional volumetric spoiled-gradient recalled echo sequence with 124 contiguous partitions, 1.6-mm partition thickness, a 22 cm × 16.5 cm field of view, 192 views, and 45° flip angle.

All image processing steps in every subject were performed by one of the authors (Y.C.X.), who was blinded to all clinical and pathologic information. The image data were interpolated inplane to the equivalent of a 512 × 512 matrix and magnified two times. The voxel size of the fully processed image data was 0.316 mm³. B₁ field inhomogeneity correction was performed. After the boundaries of the hippocampi had been delineated on each anatomic slice, the number of voxels was calculated automatically with a summing region of interest function. These were multiplied by voxel volume to give a numeric value in mm³.

The borders of the right and left hippocampi were manually traced with a mouse driven cursor for each slice sequentially from posterior to anterior. Inplane hippocampal anatomic boundaries were defined to include the CA1 through CA4 sectors of the hippocampus proper, the dentate gyrus, and subiculum.10 The posterior boundary of the hippocampus was determined by the oblique coronal anatomic section on which the crura of the fornices were identified in full profile. The intrarater test–retest reproducibility of this method has been verified with a coefficient of variation of 0.28%.17 Intracranial volume was determined by tracing the margins of the inner table of the skull on contiguous images from the sagittal sequence.

Pathology methods.

At autopsy, the brains were removed using a standard protocol. One hemisphere was sectioned in 1-cm coronal slabs. Standard histologic sections were taken, including sections of the hippocampus with adjacent inferior temporal lobe. These paraffin-embedded blocks of temporal lobe were sent to Mayo Clinic Jacksonville, where the pathologic analyses were performed by an investigator (D.W.D.) who was blinded to clinical, radiologic, and pathologic information. The sections were stained with hematoxylin and eosin (H-E) and thioflavin-S. Histopathologic features such as cerebrovascular pathology and hippocampal sclerosis were ascertained from the H-E section. Thioflavin-S sections were examined with fluorescence microscopy. The number of senile plaques was counted in three contiguous 100× fields that subtend a 2-mm² area in the entorhinal cortex. The density of plaques and tangles was also assessed in the temporal neocortical gyri, but was not recorded. Neurofibrillary tangles were counted in three 40× fields that subtend 0.125 mm². Plaques and tangles were separately recorded in various sectors of Ammon’s horn (CA4, CA2/3, CA1, and the subiculum) as well as the entorhinal cortex. Tangles were recorded in layer II neuronal clusters in the entorhinal cortex. Sections were also studied with immunohistochemical methods. All cases were stained for tau, using either PHF-1 or CP13 (both monoclonal antibodies provided by Peter Davies, Albert Einstein College of Medicine, Bronx, NY), α-synuclein, using a polyclonal antibody that has been previously characterized,18 and with a polyclonal antibody to ubiquitin that has also been previously characterized.19 Immunostaining was done in baths to assure uniformity between cases, using an avidin–biotin peroxidase method (Vector Laboratories, Burlingame, CA). The density of Lewy bodies were counted in the entorhinal cortex using the α-synuclein–stained sections and the presence of Lewy neurites in CA2/3 was also noted. The dentate fascia was examined for the presence of ubiquitin-positive inclusions that are characteristic of some frontotemporal degenerative disorders.20

The specimens were assigned one or more pathologic diagnostic categories. The pathologic diagnostic categories and criteria employed for the diagnoses were as follows:

1. Typical aging changes. The presence of agyrophilic grains was considered a feature of typical aging. Limited neurofibrillary pathology (≤Braak and Braak stage IV) could be present. Senile plaques could be present (up to 20 per 100× fields) if the plaques were diffuse amyloid deposits without cores or neuritic elements.

2. AD. This diagnosis was made according to pathologic criteria formulated by a working group of the Reagan Institute of the Alzheimer’s Association and the National Institute on Aging.2

3. Diffuse Lewy body disease (DLBD). This diagnosis was established using standard criteria.21

4. Hippocampal sclerosis (HS). This diagnosis was based upon selective neuronal loss and gliosis in CA1 and the subiculum.22

5. Frontotemporal degeneration (FTD). This diagnosis required neuronal loss and gliosis associated with laminar spongiosis in the temporal lobe and hippocampus, and ubiquitin-positive, tau-negative inclusions in the dentate fascia.23 The type of FTD defined by these criteria fits the description of the motor neuron disease–type of FTD. It was a definition that permitted an accurate pathologic diagnosis with limited tissue sampling. Given that this study was confined to the temporal lobe and not other brain sections, we chose to use a restrictive definition of FTD, but one that provided for distinctive and diagnostic histopathology that could be readily detected in the temporal lobe. We acknowledge that some forms of FTD do not have ubiquitin-positive, tau-negative inclusions in the dentate fascia, but we did not feel confident in making this diagnosis without more pathologic information because such cases may have normal hippocampi.

6. Neurofibrillary tangle degeneration (NFT). Extensive neurofibrillary pathology was present in the medial temporal lobe without senile plaques or at most a few diffuse nonneuritic type plaques.24

7. Cerebral infarction. The sections were evaluated for infarcts and hemorrhages and the lesions were histologically dated. Lesions that were acute and subacute (hours to days old) were not considered likely to have been associated with antemortem MRI findings.25

Each specimen was staged for severity of neurofibrillary pathology regardless of its pathologic diagnostic category using a modification of the method of Braak and Braak, which has been shown to have clinical validity in a previous study.26 This involves matching the topographic distribution of neurofibrillary lesions to the best-fitting published template.27 Each specimen was assigned to Braak and Braak stages 0 through 6: stage I, neurofibrillary degeneration (NFD) confined to the transentorhinal cortex (layer IV); stage II, NFD in entorhinal cortex (layer II); stage III, NFD in hippocampus (CA1 and subiculum); stage IV, NFD in temporal lobe association neocortex (mild); and stage V, NFD in temporal lobe association neocortex (moderate to severe). Braak and Braak stage VI requires an assessment of the primary visual cortex, which was not available for this study, but in stage VI there are hippocampal changes (e.g., neurofibrillary tangles in the dentate fascia) that can be used as a guide to diagnosis of end-stage disease.

Statistical methods.

MRI-based measurements were normalized for individual variation in head size, age, and sex. The measured volume of the hippocampus ipsilateral to the pathologically assessed hemisphere in each individual was divided by the total intracranial volume of that individual to control for intersubject variation in head size. We have previously determined age- and sex-specific normal percentiles for head size–normalized hippocampal volume in a group of 126 cognitively normal elderly control subjects.10 Age- and sex-specific normal percentiles for each of the patients in the current study were calculated from this normal elderly sample. Each percentile was converted to a W score. The W score is the value from a standard normal distribution corresponding to the observed percentile in controls. For example, for a standard normal distribution, the 50th, 5th, and 2.5th percentiles are given by 0, −1.64, and −1.96, respectively. Thus, an individual with a head size–normalized hippocampal volume at the 5th percentile of normal, after adjustment for age and sex, would receive a W score of −1.64. Similarly, an individual at the 50th percentile would receive a W score of 0. W scores provide a method to assess the degree to which head size–normalized hippocampal volume measurements of individual subjects deviate from that expected in normal subjects after adjustment for age and sex.

Comparison of W scores among pathologic diagnostic groups was performed using Kruskal–Wallis one-way analysis of variance (ANOVA) by ranks. Associations among hippocampal W score, Braak and Braak stage, and MMSE score were measured using Spearman rank correlations.

Single pathologic diagnoses.

The subjects were divided into two major groups: those who received a single pathologic diagnosis (n = 57, table 1), and those who received mixed pathologic diagnoses (n = 10, table 2). Subjects in table 1 can, in turn, be divided into three subgroups: those with 1) typical aging changes (n = 25), 2) AD (n = 23), and 3) pathologic diagnosis other than AD that were consistent with dementia (n = 9). Clinical diagnoses in the 25 subjects who were classified pathologically as having changes of typical aging were: cognitively normal control (10), MCI (9), and dementia (6). The six subjects clinically classified as demented but who did not have significant neurodegenerative pathology included the following: a 96-year-old woman who was untestable for MMSE; a 96-year-old man, MMSE score = 20; a 90-year-old woman, MMSE score = 22; an 82-year-old man, MMSE score = 21; an 83-year-old man, MMSE score = 15; and an 81-year-old man, MMSE score = 10.

Twenty-three subjects were classified pathologically as having AD and their clinical diagnoses were as follows: dementia (18), MCI (2), and normal control (3). The three subjects clinically classified as cognitively normal, but who received a pathologic diagnosis of AD included the following: a 102-year-old woman, MMSE score = 27, a 100-year-old woman, MMSE score = 23; and an 83-year-old woman, MMSE score = 26.

The remaining 9 subjects in table 1 had pathologic diagnoses other than AD that were consistent with dementia. Three of these were pathologically classified as hippocampal sclerosis, two as frontotemporal degeneration, and one as neurofibrillary tangle–only degeneration; all of these were clinically labeled demented. Three subjects were pathologically classified as DLBD. Two were clinically demented. The third, a 93-year-old man with an MMSE score of 24, received a clinical diagnosis of cognitively normal.

In general, the pathologic diagnosis of isolated AD was associated with a high Braak and Braak stage, low MMSE score, and a low MRI hippocampal W score (i.e., significant hippocampal atrophy relative to control subjects). In contrast, those with a histologic diagnosis of typical aging had lower Braak and Braak scores, higher hippocampal W scores (i.e., little atrophy), and higher MMSE scores.

Among the subjects in the third subgroup from table 1, isolated non-AD pathology associated with dementia, those with HS and FTD had low Braak and Braak scores, low hippocampal W scores, and low MMSE scores. The single individual with NFT degeneration had a moderate Braak and Braak score, low hippocampal W score, and low MMSE score. In contrast, although individuals with DLBD had low Braak and Braak scores as expected, two of the three had no hippocampal atrophy.

Mixed pathologic diagnoses.

The 10 subjects with mixed pathology (see table 2) can be divided into two subgroups: 1) those with mixed pathology in whom one of the pathologic diagnoses was AD (n = 7), and 2) those with mixed pathology without AD (n = 3). All subjects in table 2 were clinically diagnosed as demented except one individual who was classified as having AD plus DLBD (MMSE score = 24), who received a clinical diagnosis of MCI. The seven subjects with AD plus a second pathologic diagnosis all had high Braak and Braak scores and significant hippocampal atrophy. The three subjects with mixed pathology without AD all had low Braak and Braak scores, but still had significant hippocampal atrophy.

Correlation between hippocampal W score and pathologic diagnosis.

The median (range) hippocampal W scores by pathologic diagnostic subgroup were as follows: 1) typical aging pathology (n = 25, see table 1) −0.5 (−2.1 to 1.5), 2) AD (n = 23, see table 1) −2.1 (−2.9 to 0.5), 3) non-AD dementia pathology (n = 9, see table 1) −2.2 (−2.7 to 0.7), 4) mixed AD (n = 7, see table 2) −2.2 (−2.7 to 0.4), and 5) mixed non-AD dementia pathology (n = 3, see table 2) −2.5 (−3.0 to −1.9). The median hippocampal W scores were significantly smaller (more atrophic) in each of Subgroups 2 through 4 above compared with the typical aging subgroup (p < 0.01 vs Groups 2,4, and 5, and p = 0.02 vs Group 3). Pairwise comparisons among Subgroups 2 through 5 revealed no significant differences in hippocampal W score.

Correlation among Braak and Braak stage, hippocampal W score, and MMSE score.

When all 67 subjects in tables 1 and 2⇑ were considered together, significant correlations were present between hippocampal W score and Braak and Braak stage, hippocampal W score and MMSE, as well as MMSE and Braak Stage (table 3). Of the five individual pathologic diagnostic subgroups, only the AD (n = 23) and typical aging (n = 25) subgroups had sufficient numbers of individuals to permit within-group statistical correlations between Braak and Braak staging, hippocampal volume, and MMSE. Among patients with AD, Braak and Braak stage correlated inversely with both hippocampal W score (i.e., greater atrophy) and MMSE score (figure). Hippocampal W score was also correlated with MMSE score. The only significant correlation present in patients with typical aging was between hippocampal W score and MMSE score.

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Figure. Braak and Braak stage vs normalized bilateral hippocampal volume in subjects with isolated AD pathology.