Identifying severely atrophic cortical subregions in Alzheimer’s disease

Neurobiology of Aging 24 (2003) 797–806

Identifying severely atrophic cortical subregions in Alzheimer’s disease G.M. Halliday a,∗ , K.L. Double a , V. Macdonald a , J.J. Kril b b

a Prince of Wales Medical Research Institute, The University of New South Wales, Barker Street, Randwick 2031, Australia The Centre for Education and Research on Ageing, Departments of Medicine and Pathology, University of Sydney, Sydney 2006, Australia

Received 6 August 2001; received in revised form 14 November 2002; accepted 20 November 2002

Abstract The present study analyses the pattern of atrophy in anatomically discrete brain regions in prospectively-studied pathologically-confirmed patients with Alzheimer’s disease (AD) and controls. Standard volumetric measurements were made of the entire cortex subdivided into 23 anatomical regions. Analyses determined regions of significant atrophy in AD and differences between the severity and rates of atrophy. Two levels of severity were found. Atrophy concentrated in medial temporal lobe structures as well as in inferior temporal and superior and middle frontal cortices. The degree of atrophy in these regions related to disease duration, consistent with an early and sustained disease process. The rate of atrophy was significantly greater for the fusiform gyrus. The inferior frontal lobes were entirely spared at all AD stages, while atrophy of other cortical regions was less marked and not related to disease duration, suggesting late involvement. Our findings show that the fusiform gyrus is particularly affected by AD, and suggest two levels of atrophy that correspond with published neurofibrillary tangle (most atrophic) and senile plaque (less atrophic) densities. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Amygdala; Hippocampus; Middle and superior frontal cortical gyri; Temporal cortex; Regional volumes

1. Introduction Alzheimer’s disease (AD) is a progressive dementia characterised by degeneration and atrophy which concentrates in the temporal lobe [5,22,67]. There is considerable ongoing research effort to determine the most discriminate volumetric measurements to differentiate AD from both normal elderly subjects and patients with other pathologies [16,67]. These studies typically use a cross sectional design and, because of the laborious task to identify specific regions for volumetric analysis, only limited information is available on the progression of atrophy throughout the entire cortex and over the disease course. Knowledge of the pattern/s of cortical atrophy over time may help identify regions of consistent, early and less variable atrophy versus regions undergoing atrophy later in the disease course. The identification of such regions may provide insights into the pathogenesis of AD. Longitudinal studies have demonstrated that the rate of brain atrophy varies, depending upon the disease stage. Jobst et al. [42] reported an average annual rate of atrophy of 15% in the medial temporal lobe in autopsy-confirmed AD over the entire disease course, while studies of probable ∗

Corresponding author. Tel.: +61-2-9382-2736; fax: +61-2-9382-2681. E-mail address: [email protected] (G.M. Halliday).

AD analysing shorter time epochs suggest lower rates of hippocampal atrophy varying from 3–4 [40] to 7–10% per annum [53]. During conversion to AD, the rate of hippocampal atrophy develops a steep linear slope [28,40], indicative of the progressive nature of the disease [25,50,57,72]. Studies of short disease periods suggest that, in addition to marked hippocampal atrophy, a milder, more global cortical atrophy occurs, beginning in the inferolateral temporal, posterior cingulate and parietal areas [28]. The rate of cortical atrophy is 2–3% per annum [10,27,28], and is relatively homogeneous for different AD patients [10]. The changing pattern of regional atrophy in AD needs further assessment to clearly establish regional atrophy heralding the disease versus regions that atrophy at symptom onset and regions that atrophy following diagnosis. Neuropathological studies of cross sectional design have provided valuable information concerning the progression of pathology from the elderly without dementia to those with definite AD. By comparing cases and controls across different disease stages the temporal pattern of pathology has been determined [7,21,24,57,72]. The relative invariance in the temporal sequence of pathology shows a stable disease progression and decline, validating clinical studies [12,30,55,58]. In clinical studies the rate of cognitive decline appears similar over time (±2 years) in patients with ‘pure’ AD [30], and is consistent with rates of atrophy [10].

0197-4580/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(02)00227-0

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The analysis of pathologically homogeneous cross sectional cohorts of variable disease duration can be used to determine the relative rates of decline in anatomical subregions of the brain. The present study analyses the degree of regional brain atrophy across the entire cortex (similar to [70]) in a cohort of AD patients with variable disease duration in comparison to age- and sex-matched controls. All major gyri and structures in frontal, temporal and parietal cortices were analysed to ensure confirmation of diagnosis and determine atrophy caused by AD in isolation. 2. Materials and methods

(CDR = 0) and were without significant macroscopic or microscopic neuropathological abnormalities. Age of onset of AD was judged by the patient’s neurologist on the basis of informant history at initial clinical interview. Of note, 62% of cases had clinical features for 4 years or less at recruitment (average duration = 2 ± 1 years). Average disease duration (±S.D.) was 9 ± 3 years (range: 4–14 years, females: 8.6 ± 3.5 years, males: 9.4 ± 3.4 years). Except for two AD cases, all had severe end-stage dementia (CDR = 4–5) and were in nursing homes at the time of death. The two non-end stage cases had moderate to severe dementia (CDR = 2–3). The postmortem interval was 38 h or less in all cases (range; 4–38 h, mean ± S.D. for controls = 19 ± 8 and for AD = 17 ± 11 h, t-test = 0.8, P = 0.50).

2.1. Case selection All subjects were prospectively studied and some have been reported previously [22,35]. The subjects were participants in a case-control study of AD in an urban population [9] prior to enrolling in our brain donor program. The sample consisted of newly recognised cases of dementia living in the community near two adjacent Sydney hospitals (Concord Repatriation General and Lidcombe Hospitals) recruited through general practitioners over a 2.5-year period. Clinical diagnosis of AD was made according to NINCDS-ADRDA [51] criteria and all donors were evaluated yearly until the time of death. Progression of clinical dementia was assessed using the clinical dementia rating (CDR) score [54]. Fifty percent agreed to genotyping for apolipoprotein E. The majority of cases and controls had ε3/3 genotype (85–88%), with the remaining having either 3/4 or 4/4 genotypes. Questionnaires sent to relatives and general practitioners at the time of death were used to verify cognitive status, including the presence and severity of dementia using the CDR. Patients who significantly declined between their last clinical assessment and death were excluded. The study was approved by the Human Ethics Committees of the South Eastern and Central Sydney Areas Health Services and The Universities of Sydney and New South Wales and complies with the statement on human experimentation issued by the National Health and Medical Research Council of Australia. Standard neuropathological research criteria were used to exclude cases with degenerative disorders other than AD [11,36,69]. Seventeen cases of AD (nine males, eight females; age range: 66–92 years) and 21 controls (11 males, 10 females; age range: 65–92 years) were chosen for evaluation. Cases and controls were matched for age (mean ± S.D. for controls = 75 ± 11 years old and for AD = 77 ± 8 years old, t test = 0.9, P = 0.37) and apolipoprotein E genotype (88% controls had ε3/3 genotype and 85% AD had ε3/3 apolipoprotein E genotype). The average interval ± S.D. between the last clinical assessment and death was 10 ± 7 months. Cases with AD fulfilled CERAD criteria for probable or definite AD [52] and had stage 4, 5 or 6 neuritic pathology [6]. Controls had no evidence of dementia

2.2. Brain preparation and identification of regions of interest Each brain was weighed at autopsy and the volume determined by fluid displacement. Following fixation for 14 days in 15% neutral buffered formalin, the weight and volume was remeasured to determine formalin-induced shrinkage. As previously described [22,35], shrinkage was minimal in these cases using this protocol (0.7 ± 0.7% change from fresh volume). The cerebellum and brainstem were separated from the cerebrum by sectioning through the cerebral peduncles. The weight and volume of the cerebrum was determined and the length of each hemisphere measured using large calipers and a metric ruler. Each cerebrum was embedded in 3% agarose, sectioned in 3 mm coronal slices, photographed and printed (magnification: 1×). The entire cortical volume was measured in all cases. To aid in the identification of regions once coronally sliced, different cortical gyri were painted with coloured dyes prior to sectioning, as previously reported [34,44,63]. Using the painted cortical surfaces as landmarks, anatomical structures and gyral boundaries were identified which consistently distinguished the regions of interest according to the method of Harasty et al. [34]. This method uses gyral boundaries and deep brain structures consistently associated with the gyral boundaries to identify the same regions across all cases. The gyral boundaries identified (Fig. 1) are in accordance with human brain atlases [15,49] and are similar to the cortical regions recently measured in vivo by Thompson et al. [70]. Anatomical descriptions of the cortical regions of interest include reference to Brodmann’s map for standardised location, rather than implying strict cytoarchitectural similarity. 2.2.1. Limbic regions 2.2.1.1. Amygdala. It is defined in each slice using boundaries of de Olmos [17]. The amygdala is an almond shaped grey matter structure in the anteromedial temporal lobe just posterior to where the temporal pole fuses to the rest of the brain. Its medial surface is the medial surface of the temporal lobe, its lateral surface is the temporal horn of the

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Fig. 1. Photos and diagrams of the external brain features prior to sectioning (A, B) and after coronal slicing (C, D) of a representative control (A, C) and AD case (B, D). The different cortical regions were identified using the main sulcal patterns of the brain (A, B) and the consistent positioning of internal brain structures in the coronal slices (C, D) (see Section 2). In this way different cortical gyri and regions could be accurately identified in all cases despite gross morphological differences (compare A with B).

lateral ventricle, and its anterior and superior boundaries are formed by white matter tracts. Its inferior border is defined by the uncal notch and separates it both inferiorly and posteriorly from the entorhinal cortex (inferiorly) and the anterior hippocampus (posteroinferiorly). 2.2.1.2. Hippocampus. It is defined in each slice using boundaries of Amaral and Insausti [1]. The medial surface of the hippocampus is the medial surface of the temporal lobe, its lateral and superior borders are the temporal horn of the lateral ventricle, and its inferior border is the entorhinal and posterior parahippocampal cortices and underlying white matter. Anterosuperiorly it abuts the amygdala and its

posterior border is the crux of the fornix. The anteroposterior extent of the hippocampus has been verified histologically in a subset of cases [37]. 2.2.1.3. Entorhinal cortex. It is defined in each slice using boundaries of Insausti et al. [39]and Krimer et al. [45] (Brodmann’s area 28). The medial and inferior surfaces of the entorhinal cortex are the medial and inferior surfaces of the temporal lobe, its lateral border is the depth of the collateral sulcus, and its superior border is the hippocampus and underlying white matter. For standardization, its anterior boundary was defined by fusion of the temporal pole to the rest of the brain, in keeping with histopathological

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studies of this region [38]. The posterior boundary occurred at the level of the lateral geniculate nucleus, as we and others have shown [1,38,39]. 2.2.1.4. Posterior parahippocampal cortex. Parahippocampal gyri posterior to entorhinal cortex (Brodmann’s areas 28, 35). The posterior limit was defined as the last slice containing the related hippocampi. 2.2.1.5. Anterior cingulate gyri. Cortex of the cingulate gyrus (above the corpus callosum and limited by the cingulate and subparietal sulci) with the posterior limit at the medial level of the central sulcus (Brodmann’s areas 24, 32). 2.2.1.6. Posterior cingulate gyri. Cortex of the cingulate gyrus (above the corpus callosum and limited by the cingulate and subparietal sulci) with the anterior limit at the medial level of the central sulcus and the posterior limit the end of the cingulate gyrus (Brodmann’s areas 23, 31). 2.2.2. Temporal lobe regions—posterior extent taken to the last coronal slice containing the most posterior aspect of the hippocampus 2.2.2.1. Anterior temporal pole. All temporal cortex anterior to the insula (Brodmann’s area 38), that is all temporal cortex in the coronal slices of the temporal lobe not attached to the rest of the cerebrum. 2.2.2.2. Fusiform gyri. Gyrus limited by the collateral and temperooccipital sulci (Brodmann’s areas 20, 36). The anterior limit was taken as the first appearance of the gyrus between the parahippocampal and inferior temporal gyri posterior to the temporal pole, and the posterior limit was the most posterior extent of the Sylvian sulci. 2.2.2.3. Inferior temporal gyri. Gyrus limited by the temperooccipital and inferior temporal sulci (Brodmann’s area 20). The anterior limit was taken as the coronal slice of the temporal lobe attached to the rest of the cerebrum, and the posterior limit was the most posterior extent of the Sylvian sulci. 2.2.2.4. Middle temporal gyri. Gyrus limited by the inferior and superior temporal sulci (Brodmann’s area 21). The anterior limit was taken as the coronal slice of the temporal lobe attached to the rest of the cerebrum, and the posterior limit was the most posterior extent of the Sylvian sulci.

2.2.3. Frontal lobe regions—posterior extent taken to the central sulci 2.2.3.1. Frontal pole. All cortex in the slices anterior to the commencement of the anterior cingulate gyrus (Brodmann’s area 10). 2.2.3.2. Orbitofrontal cortex. Cortex inferior to the corpus callosum, excluding the anterior cingulate gyrus (Brodmann’s areas 11, 25). 2.2.3.3. Inferior frontal gyri. Posterior inferior frontal gyri defined by the Sylvian fissure and inferior frontal sulci (Brodmann’s areas 44, 45, 47). 2.2.3.4. Superior and middle frontal gyri. Posterior superior and middle frontal gyri defined by the inferior frontal and cingulate sulci (Brodmann’s areas 8, 9, 46). 2.2.3.5. Motor cortices. Gyri anterior and parallel to the central sulcus and posterior to the precentral sulci (Brodmann’s areas 4, 6). 2.2.4. Parietal lobe regions—anterior boundary from the central sulci, posterior boundary the parietooccipital fissure 2.2.4.1. Somatosensory cortex. Gyri defined as posterior to the central sulci and anterior to the postcentral sulci (Brodmann’s areas 1, 2, 3, 5). 2.2.4.2. Superior parietal lobule. Cortex posterior to the postcentral sulci, anterior to the parietooccipital notch and superior to the intraparietal sulci (Brodmann’s area 7). 2.2.4.3. Supramarginal gyri. Cortex defined by the postcentral sulci anteriorly, the intraparietal sulci superiorly, the Sylvian sulci inferiorly and the posterior ascending rami of the Sylvian sulci posteriorly (Brodmann’s area 40). 2.2.4.4. Angular gyri. Cortex defined as posterior to the posterior portion of the superior temporal gyri and the Sylvian sulci, inferior to the intraparietal sulci and extending to occipital cortex (Brodmann’s area 39). 2.2.4.5. Area 37. Cortex inferior to angular gyri, posterior to temporal lobe regions and defined posteriorly by the anterior occipital sulci (Brodmann’s area 37). 2.2.5. Other regions

2.2.2.5. Superior temporal gyri. Gyrus limited by the Sylvian fissure and superior temporal sulci (Brodmann’s areas 22, 41, 42). The anterior limit was taken as the coronal slice of the temporal lobe attached to the rest of the cerebrum, and the posterior limit was the most posterior extent of the Sylvian sulci.

2.2.5.1. Insula. Cortex in the depths of the Sylvian sulci. The anterior limit was defined as the first slice containing the adherence of the temporal pole to the rest of the cerebrum and the posterior limit was the most posterior extent of the Sylvian sulci.

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2.2.5.2. Occipital lobe. Cortex defined from the parietooccipital notch, posterior to the splenium of the corpus callosum and the anterior limit of the calcarine fissure (Brodmann’s areas 17, 18, 19). 2.3. Volume determination The volumes of the regions of interest were determined by a point counting procedure. All regions of interest in each brain slice photograph were identified. The slice photograph was randomly overlayed with a grid of 3848 points (area: 286 mm × 196 mm), and the number of points falling on each region identified, and the total number of points within each brain slice counted. The volume of each region was calculated by multiplying the sum of the points falling on a given structure by the volume represented by each point (volume/point = total number of points counted/cerebrum volume; average of 0.05 ml). This method is routinely used in our laboratory to measure regional volumes postmortem [14,22,23,33,35,44] and approximates current point counting procedures used in MRI studies of brain volumes. 2.4. Statistical analysis Four raters were used to identify regions of interest and determine their volume. These raters were initially trained to identify the same regions of interest in four brains with high point-to-point agreement (of the 23 cortical regions identified, agreement was perfect for two of the four raters and near perfect for the other two raters, i.e. point-to-point agreement [59] was 0.99). A coronal slice through the amygdala was chosen from these four brains for rater training in the point counting technique. The rater was considered competent at this technique when less than 5% variation was obtained for all regions of interest within this slice for each of the four brains. All raters (N = 4) were required to count all regions of interest in one male control brain and one female AD brain in order to determine inter-rater reliability using intraclass correlation. The coefficient of error for each regional volume measurement for each group was calculated as an indicator of accuracy. The lower this value is, the more accurately the mean value attained reflects the true mean. It is known that age, sex, size and laterality affect brain volumes. Average cerebrum volume declines with advancing age and males have on average significantly larger brains than females [20]. As we and others have previously described [14,19,20,22,35,56], the variance due to gender at any age (∼200 ml or 15% of the average cerebrum volume) is significantly greater than the yearly decline in volume (∼6 ml per year or 0.5% per year of the average cerebrum volume). Many previous in vivo three-dimensional volumetric studies have used intracranial volume to correct for individual size differences. As with other postmortem morphometric studies, intracranial volumes were not available to normalise our data for head size. Therefore, to control for age and individual cerebral size, the samples were

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age-matched and the variance in cerebrum volume was ascertained as similar for each group (10–15% variance). As regional volumes for these variables were not standardised, only substantial disease effects will be seen. The data were analysed by repeated measure analysis of variance (ANOVA), with diagnosis and gender as between subject factors and hemisphere as a within subject factor. One sample Kolmogorov–Smirnov tests demonstrated the data to be normally distributed for each gender-diagnosis comparison group for each measure (P > 0.05). Analyses with P values of less than 0.05 were assessed posthoc using individual t tests with Bonferroni correction for multiple comparisons to confirm that diagnosis was a significant factor influencing regional volumes. The mean ± S.D. are given for all variables. Due to the gender differences, standardization of hemispheric volumes in each gender was performed to assess the disease effect on each region. Mean control values for each hemispheric region of interest for each gender were calculated and the percentage of the control volume calculated for each AD case. To determine if the amount of atrophy differed in severity between different regions, a Kruskal–Wallis ranking test was used with posthoc Mann–Whitney U-tests to determine which regions differed from each other. Three levels of severity were found and Kruskal–Wallis ranking tests were used to verify that regions of similar severity did not differ from each other. To identify any relationships between regional cortical atrophy in each hemisphere and disease duration, these variables were submitted to a common factor analysis (20 variables with 34 datapoints each). Variables with standard coefficient loadings < 0.65 were not considered significant. Regression plots were performed for related variables to understand these relationships and differences in the regression coefficients tested using F-distribution with disease duration as the co-variate. 3. Results 3.1. Measurement accuracy and reliability Intraclass correlation coefficient for subregional volume measurements by four raters for the control was 0.959 and for the AD brain was 0.967. This indicates a high reproducibility and reliability in the identification and measurement of the subregions chosen. The coefficient of error for each regional volume measure was <0.05 (range: 0.021–0.048; average: 0.032 ± 0.008). This confirms that the volume method used is highly reliable and reproducible for the subregions identified. 3.2. Regional atrophy in AD 3.2.1. Limbic regions As expected from the literature, most but not all limbic regions were atrophic in AD (Table 1). The posterior parahippocampal gyrus remained unaffected with atrophy

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Table 1 The average regional volumes (±S.E.) and P values for the statistical analysis (ANOVA using diagnosis, gender and hemisphere as variables) Control (ml)

AD (ml)

Diagnosis

Gender

Hemisphere

Limbic regions Amygdala Hippocampus Entorhinal Posterior parahippocampus Posterior cingulate Anterior cingulate

1.72 4.7 1.91 1.43 4.7 6.1

± ± ± ± ± ±

0.05 0.1 0.09 0.06 0.2 0.2

1.12 3.0 1.15 1.38 3.5 4.7

± ± ± ± ± ±

0.06 0.1 0.06 0.13 0.2 0.2

<0.0001 <0.0001 <0.0001 0.76 <0.0001 <0.0001

0.16 0.0002 (17) 0.81 0.73 0.20 0.15

0.85 0.67 0.65 0.34 0.46 0.10

Temporal cortex Temporal pole Fusiform Inferior temporal Middle temporal Superior temporal

11.1 3.4 6.6 8.5 11.2

± ± ± ± ±

0.4 0.1 0.2 0.2 0.3

6.9 2.2 4.4 6.0 8.3

± ± ± ± ±

0.4 0.2 0.2 0.2 0.3

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001

0.007 (21) 0.0002 (18) 0.06 0.02 (11) 0.01 (7)

0.74 0.88 0.93 0.87 0.14

Parietal cortex Area 37 Angular Supramarginal Superior parietal Postcentral

10.9 14.3 12.6 18.9 11.7

± ± ± ± ±

0.4 0.4 0.3 0.6 0.3

8.7 9.6 10.1 15.1 11.0

± ± ± ± ±

0.7 0.7 0.4 0.5 0.4

0.002 <0.0001 <0.0001 <0.0001 0.15

0.22 0.92 0.08 0.34 0.40

0.18 0.98 0.64 0.43 0.77

Frontal cortex Frontal pole Orbitofrontal Inferior frontal Superior/middle frontal Motor cortices

42 10.2 5.8 14.2 19.7

± ± ± ± ±

1 0.4 0.3 0.4 0.4

33 9.7 6.0 8.9 15.4

± ± ± ± ±

1 0.4 0.3 0.7 0.6

<0.0001 0.13 0.12 <0.0001 <0.0001

0.17 0.48 0.86 0.002 (7) 0.02 (6)

0.39 0.65 0.76 0.91 0.55

3.3 ± 0.1 75 ± 3

<0.0001 0.003

0.12 0.02 (14)

0.86 0.84

Other cortices Insula Occipital

4.2 ± 0.1 87 ± 2

There were no significant interactions between variables for any regional volume. The percentage difference between male and females is given in brackets for gender.

concentrating in the entorhinal cortex, amygdala and hippocampus (Table 1). These regions were disproportionately atrophic when analysed as a proportion of cerebrum volume for each individual. Cingulate cortices were also significantly atrophic, but not disproportionately (Table 1). Of these regions, a gender difference was found for the volume of the hippocampus only (males larger than females, Table 1). There was no diagnosis by gender interaction (P = 0.96) indicating that a similar degree of hippocampal atrophy occurs in both genders. No differences in the volumes between hemispheres were found (Table 1). An average 32% atrophy of limbic regions occurred in the AD cases compared with controls. 3.2.2. Temporal cortices All temporal cortices were atrophic in the AD cases (Table 1). The temporal cortices were similar in volume across hemispheres but significantly larger in males compared with females (Table 1). The degree of temporal atrophy in AD was similar to that observed for limbic brain regions (average of 33% atrophy, Table 1) with no diagnosis by gender interactions (P values > 0.14). The greatest atrophy occurred in the temporal pole, fusiform and infe-

rior temporal gyri (Table 1), regions closest to the atrophic limbic regions identified above, but all temporal cortices were disproportionately atrophic in AD. 3.2.3. Parietal cortices The majority of the parietal lobe was also atrophic in AD with only the postcentral gyrus unaffected by the disease process (Table 1). No gender or side differences were found in the volumes of the parietal cortices (Table 1). An average 22% atrophy occurred across the parietal cortices, concentrating in area 37 and the angular gyrus (Table 1). These regions were disproportionately atrophic being located posteriorly and inferiorly within the parietal lobe, closest to the atrophic temporal cortices described above. 3.2.4. Frontal cortices Frontal cortices were similar in volume across hemispheres with superior frontal regions significantly larger in males compared with females (Table 1). Significant atrophy occurred in the anterior and superior frontal cortices, including the motor cortices (Table 1). These regions atrophied on average by 27% in AD compared with controls, largely because of the considerable atrophy in the superior and mid-

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dle frontal gyri (average 38% atrophy, Table 1). This frontal region was disproportionately atrophic when volumes were corrected for brain size. No atrophy was observed in either the orbitofrontal or inferior frontal cortices (Table 1). There were no diagnosis by gender interactions (P values > 0.06). 3.2.5. Other cortices Both the insula and occipital cortices were atrophic in AD (Table 1). There was no difference in volume between hemispheres for either region or between genders for the insula (Table 1). The occipital cortex in males was significantly larger than that measured in females (Table 1) with no diagnosis by gender interaction (P = 0.44). In the AD cases, the occipital cortex had an average atrophy of 20% for both genders, whereas the insula was atrophic by only 12% compared with controls. 3.3. Severity of regional atrophy To determine whether the atrophy described above significantly differed between regions, a Kruskal–Wallis rank test was used followed by posthoc ranking tests. This analysis reveals that significant differences in the degree of atrophy between regions did occur (P < 0.0001). Posthoc testing revealed three levels of atrophy—severely atrophic, moderately atrophic and unaffected regions. Unaffected regions were those previously identified (orbital and inferior frontal gyri, postcentral gyri, and the posterior parahippocampus, Table 1). The most severely atrophic regions were the fusiform and inferior temporal gyri, the temporal pole, the superior and middle frontal gyri and, as expected from the literature, the amygdala, hippocampus and entorhinal cortex (Fig. 2). A Kruskal–Wallis rank test confirmed that there was no difference between the degree of atrophy for these regions (P = 0.24). The remaining cortical regions were moderately atrophic (Fig. 2) and did not differ from each other using a Kruskal–Wallis rank test (P = 0.14) but differed from both the severely atrophic and unaffected groups (P < 0.001). 3.4. Relationship to disease duration Factor analysis was used to determine any relationship between disease duration and cortical atrophy. The analysis identified five factors with only two accounting for any considerable variance. Factor one accounted for 39% of the variance and was the only factor related to disease duration. This factor incorporated many of the most severely atrophic cortical regions. Atrophy of the inferior temporal (0.85), superior and middle frontal (0.77), hippocampus (0.75), fusiform (0.72), and entorhinal (0.68) gyri were all significantly related to disease duration (−0.75). Of the moderately atrophic regions, only the superior temporal gyrus (0.86) was related to this factor. Regression analysis confirmed these findings (P < 0.005, Fig. 2), and found that the slope of the regression line was the same for all

Fig. 2. Graphs of the severity of atrophy (A) and its relationship to disease duration (B). Front: frontal, hippo: hippocampus, inf: inferior, mid: middle, post: posterior, sup: superior, temp: temporal. (A) Using a Kruskal–Wallis test, significant differences between the degree of regional atrophy were found (P < 0.0001). Posthoc testing revealed three levels of change as indicated—severe, moderate or unaffected (not shown) regions. (B) Using factor and regression analyses, significant relationships were found between disease duration and atrophy of the inferior temporal, superior and middle frontal, hippocampus, fusiform, and entorhinal gyri. The regression plots show this relationship across the AD cases with the average degree of atrophy for each region at any given duration time depicted. Comparing across the same cases, the fusiform gyrus had a greater rate of atrophy than the other regions analysed (P < 0.05).

the gyri identified by factor 1, except for the fusiform gyrus which had a significantly steeper slope (P < 0.05, Fig. 2). The second factor accounted for 15% of the variance and was unrelated to disease duration. This factor identified a relationship between the volume of area 37 (0.75) and the posterior cingulate gyrus (0.71). The remaining four factors each accounted for <10% of the variance with no regional volumes gaining significant loading scores for these factors.

4. Discussion The present study mapped the pattern of cortical atrophy in a sample of pathologically-proven AD cases and demonstrated that severe atrophy occurs in limbic regions, as expected, but also in temporal and frontal cortices. These

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regions correspond to the regions of greatest tau deposition at early dementia stages [7,21]. Analyzing the degree of atrophy in cases with a spectrum of disease durations revealed that atrophy of these regions is relentlessly progressive. Additional cortical regions of atrophy are clearly separable quantitatively and were not related to disease duration. This suggests two levels of involvement with many cortical regions only consistently affected much later in the disease. The identification of this temporal pattern of atrophy in pathologically proven cases with AD is consistent with longitudinal studies of atrophy in the cortex [10,27,28,66]. While a number of studies have evaluated the topographical distribution of pathology in AD, few postmortem studies have analysed the distribution of atrophy in a similar fashion. A recent review of diagnostic neuroimaging for AD [67] emphasised both the importance of histopathological validation of atrophy and the lack of available data in this area. Confirmation of the clinical diagnosis is only possible at postmortem, and atrophic changes at this stage are likely to identify consistent patterns of atrophy. In vivo studies of clinical AD show consistent early atrophy of medial temporal lobe structures [18,41,71]. Temporal and parietal cortical regions are also considered selectively affected in AD [18], although many in vivo studies show early atrophy of frontal cortical areas [46,47,65,70]. The results of the present study confirm a distribution of atrophy incorporating the entire temporal lobe, the posterior and inferior parietal lobe, and the anterior and superior frontal lobe. At postmortem, large left/right volume differences were absent and the same pattern of atrophy was observed regardless of gender. Somewhat of a surprise was the extreme atrophy observed in several cortical association regions in addition to the amygdala, hippocampus and entorhinal cortex. In particular, the temporal pole, fusiform and inferior temporal gyri, and the middle and superior frontal gyri were as severely affected as these limbic brain structures. Atrophy of these limbic and association regions negatively correlated with disease duration with the fusiform gyrus having a significantly greater rate of atrophy than the other gyri examined. It has previously been shown that the rate of hippocampal atrophy matches cognitive decline [40], and that the rate of temporal lobe atrophy is significantly greater than that of the hippocampus [43]. This is particularly evident as the disease progresses [66]. Further, atrophy of the fusiform, inferior and middle temporal gyri accurately predict the onset of AD, with the fusiform gyrus independently predicting the disorder [12,13]. These temporal regions of the cortex appear the first to develop neurofibrillary tangles and mature neuritic plaques [2,4,25,31,62]. In addition, a 20–30% atrophy of the middle frontal gyri has recently been observed in AD patients presenting with mild deficits [70], consistent with the frontal atrophy noted in the disease [46,47]. In longitudinal studies, significant atrophy of small regions of frontal cortex are found in people prior to dementia onset, with atrophy increasing as disease progresses [66]. Glucose deficits in these frontotemporal

regions have been identified preclinically [64] and preclinical neuropsychological testing shows that disturbances in central control processes independently predict AD [26], supporting an early contribution from frontal regions [61]. Significant plaque formation but less neurofibrillary tangle formation are found in the superior frontal lobe compared with the temporal lobe [2,4,32] with the superior frontal gyrus most commonly sampled in diagnostic biopsies [3]. The concentration of plaques in both frontal and temporal cortical regions is double that found in age-matched non-demented controls [2,4,32]. Overall, the data show that selective frontal and temporal association cortical regions are affected to a similar magnitude to that observed in limbic brain regions with regression analyses suggesting that all these regions are affected early. Comparison with histopathological studies indicate that the underlying disease mechanisms may differ (plaque in superior frontal regions versus more neuritic cellular pathology in temporal cortices). Atrophy of the severely affected cortical regions related to disease duration with a relentlessly progressive profile observed when the AD group was analysed as a whole. This suggests that cases with confirmed AD pathology have a temporally similar pattern of atrophy in addition to their similar pathological profile. The majority of brain regions were involved in the disease, consistent with current knowledge of end stage pathology [8,68]. Atrophy of many of these regions was not related to disease duration, but most atrophic regions are connected to those severely affected. The most consistent and severe histopathological change in these regions is plaque deposition compared with more variable neurofibillary tangle formation [2]. However, there is a surprising absence of atrophy in the inferior frontal lobes in AD. Including measurements of these regions into volumetric paradigms for diagnosis may provide the necessary within person comparison to differentiate AD from frontotemporal dementia and/or dementia with Lewy bodies, as the frontal lobes are more consistently affected in these dementia syndromes [14,23,29]. Comparison with the proportional regional atrophy identified in vivo [70] revealed a statistically identical pattern of atrophy at postmortem. Early in the disease, there is significant atrophy of the middle frontal gyrus and temporal and parietal cortices, with sparing of occipital and sensory cortices. Sensory cortices continued to be spared at all disease stages, but the occipital cortex becomes atrophic, consistent with its pathological involvement [48] and use in disease staging [6]. The significantly greater left versus right atrophy identified in vivo [66,70] was not present postmortem. As the asymmetrical atrophy of some regions was considerable in this early in vivo study (20–30% differences) [70], it suggests that there is only a relatively small delay between interhemispheric disease progression of selectively affected association cortical regions. In support of this concept, left/right differences in hippocampal atrophy are only found in cases of questionable dementia [71] and the pat-

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tern of callosal atrophy has been shown to correlate with the regional brain atrophy observed in AD with the suggestion that interhemispheric disconnection contributes to the syndrome [60]. Overall this study has identified, in pathologicallyconfirmed cases, regional susceptibility of cortical damage in AD. The finding that atrophy occurs in functionally-discrete cortical regions in two stages, while sparing other regions, provides additional evidence for a precise and reproducible pattern of neurodegeneration in AD. Comparison with histopathological studies suggests that the relentless infiltration of neurofibrillary tangles within certain brain regions correlates with the most severe regions of atrophy with secondary and milder atrophy relating more to plaque formation.

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Acknowledgments [19]

The work was funded by the National Health and Medical Research Council of Australia and the Medical Foundation of the University of Sydney. We thank our laboratory staff for assistance with the preparation of specimens for analysis and Professor Tony Broe and Drs. Helen Creasey and Liz McCusker for clinical case assessments.

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