Neurofibrillary pathology — correlation with hippocampal formation atrophy in Alzheimer disease

Neurobiologyof Aging, Vol. 17, No. 6, pp. 909-919, 1996 Copyright © 1996 ElsevierScienceInc. Printed in the USA. AUrights reserved 0197-4580/96 $15.00 + .00 ELSEVIER

S0197-4580(96)00160-1

Neurofibrillary Pathology Correlation With Hippocampal Formation Atrophy in Alzheimer Disease M A C I E J B O B I N S K / , * J E R Z Y W E G I E L , * H E N R Y K M. W I S N I E W S K I , .1 M I C H A L T A R N A W S K I , * M A R G A R E T B O B I N S K I , * B A R R Y R E I S B E R G , t M O N Y J. D E LEONe" A N D D O U G L A S C. M I L L E R S

*New York State Institute for Basic Research in Developmental Disabilities, Department of Pathological Neurobiology, 1050 Forest Hill Road, Staten Island, NY 10314 USA "pNew York Universir; Medical Center, Aging and Dementia Research Center, Department of Psychiatry, 550 First Avenue, New York, NY 10016 USA SNew York University Medical Center, Aging and Dementia Research Center, Department of Pathology, 550 First Avenue, New York, NY 10016 USA R e c e i v e d 17 July 1995; A c c e p t e d 19 M a r c h 1996 BOBINSKI, M., J. WEGIEL, H.M. WISNIEWSKI, M. TARNAWSKI, M. BOBINSKI, B. REISBERG, M.J. DE LEON AND D, C. MILLER. Ne~rofibrillarypathology--Correlation with hippocampalformation atrophy in Alzheimerdisease. NEUROBIOL AGING 17(6) 909-919, 1996.--The three-dimensionally reconstructed hippocampal formations in three patients with very severe, immobile Alzheimer disease (AD) and three age-matched nondemented individuals were examined for a correlation between atrophy of hippocampal forraation subdivisions and neurofibrillary changes, neuronal loss, and extent of amyloid deposition in plaques and vessels. In AD, a similar severe volume loss was observed in both cellular layers and layers composed of fibers. A strong correlation between the decrea.,;e in the volume of hippocampal formation subdivisions and the decrease in the total number of neurons suggests a causative ro]~efor neuronal loss in hippocampal formation volumetric loss. Strong regional correlations between the relative decreases in the total number of neurons and the relative increases in the total number of neurofibrillary tangles implicates neurofibrillary pathology as a possible etiologic proximate factor in neuronal and volumetric loss in the hippocampal formation of AD patients. Copyrighr©1996 by ElsevierScienceInc. Hippocampal formation Morphometry

Alzheimer disease

Neurofibrillary pathology

Amyloid deposits

Neuronal loss

loss, and amyloid deposition in plaques and vascular walls (4,5, 11,12). Neurofibrillary degeneration and the loss of projection neurons responsible for the majority of afferent and efferent connections of the hippocampal formation cause both the disruption of intrahippocampal connections and functional isolation of the hippocampal formation from other parts of the memory system. Neuronal loss in the hippocampal formation appears to be a major component of the memory impairment seen in AD (34). Neurofibrillary degeneration has been put forth as a cause of neuronal loss and atrophy of the hippocampal formation (3,18,34,43). Later onset of amyloid deposits in hippocampal formation subdivisions and topographical differences in distribution and number suggest that amyloid and neurofibrillary changes are most likely unrelated and develop independently (9,13). The impact of amyloid deposits on hippocampal formation atrophy is not known. Commonly used morphometric measures of AD pathology in the hippocampal formation are numerical density of neurons and

IMPAIRMENT of memol~y is one of the earliest features of Alzheimer disease (AD). Clinical symptoms slowly increase in severity and are accompanied by personality changes, deterioration of language functions, and involvement of the extrapyramidal motor system (48). Disease sew~rity is associated with a hierarchy of pathologic changes in the entorhinal cortex, hippocampal formation, and the isocortex (12). Nearly one-third of clinically normal subjects older than 55 ye~:s show mild psychometrically manifest memory impairment and evidence of hippocampal atrophy on radiographic examination (27). The prevalence of hippocampal atrophy increases with age (19,27,36,51). MRI (17,36,37) and postmortem histopathological studies show that the hippocampal formation is one of the earliest and most affected structures in AD. Atrophy of the hippocampal formation subdivisions correlates with the stage and duration of AD (8). Morphological studies of the hippocampal formation reveal neurofibrillary changes and granulovacuolar degeneration of neurons, synaptic and neuronal

1 Requests for reprints should be addressed to Dr. Henryk Wisniewski, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399. 909

910

BOBINSKI ET AL.

neurofibrillary tangles (NFTs) (3,18,34,43). However, total number of neurons is considered a better measure of the functional capacity of the structure than numerical density (46,55,57). The aim of this study was to examine atrophy of the hippocampal formation subdivisions and its possible correlation with neurofibrillary changes, neuronal loss, and extent of amyloid deposition in various types of plaques and vessels. METHOD One hippocampal formation from the brains of each of three subjects with very severe AD (a 77-year-old female and males 77 and 86 years of age) and of three control subjects with no clinical dementia (a 71-year-old female and 79- and 82-year-old males) was reconstructed and examined morphometrically. The nondemented control group consisted of subjects who lived independently, had no history of neurological disease and died of pneumonia (two cases) and hepatitis. Dementia of AD patients was assessed by the Global Deterioration Scale (GDS) (48) as GDS stage 7, and by the Functional Assessment Staging procedure (FAST) (47) as FAST stage 7e. The duration of AD in these nonverbal, nonarnbulatory, immobile AD patients who could no longer sit up independently, and were additionally evaluated as having lost the ability to smile, was assessed using procedures described previously (8). The duration ranged from 19 to 22 years from the onset of clinically manifest symptoms in GDS and FAST stage 3, until demise. After at least 6 weeks of fixation in 10% buffered formalin, brains were divided sagittally and one hemisphere was cut coronally into 4.8-mm thick slabs, processed, and en,'~edded in paraffin. Paraffin blocks containing the hippocampal formation were cut serially into 8-txm thick sections. Every 50th section was stained with cresyl violet and used for planimetry. Two adjacent sections from each slab were stained, respectively, with monoclonal antibody (mAb) tau-1 raised against abnormally phosphorylated tau, and detecting an epitope between amino acids 189 and 207 of the human tan sequence (26), or with mAb 4G8, which detects an epitope between amino acids 17 to 24 of the arnyloid [3 protein. Sections were pretreated with alkaline phosphatase (Sigma, Type VII-L, 400 Ixg/ml in PBS, pH 7.4, 0.01% H202) for mAb tau-1 immunostaining(28) and with concentrated formic acid for 30 min to enhance immunoreactivity of amyloid (39). After immunostaining, sections were counterstained with cresyl violet. The clinical diagnosis of AD was confirmed histopathologically according to the age-adjusted guidelines suggested by the National Institute on Aging (38).

Planimetry and Volumetry of the Hippocampal Formation The area of the hippocampal formation and its major subdivisions was measured on the serial sections. Division of the cornu Ammonis, dentate gyrus, and subicular complex into layers and sectors was based on anatomical and cytoarchitectural criteria of Lorento de No (40), Braak (10), Duvernoy (22), Amaral and Insausti (1), and Rosene and Van Hoesen (49). The alveus, strata oriens, pyramidale, radiatum, and lacunosum/moleculare were distinguished in sectors CA1 to CA3 of the cornu Ammonis and the stratum pyramidale in the CA4 sector. The area of the molecular, granular, and polymorphic layers was measured in the dentate gyrus. In the subiculum proper and in the parasubiculum, the molecular and pyramidal layers were examined, whereas in the presubiculum, the molecular, parvopyramidal, and pyramidal layers were assessed. The entorhinal cortex was examined planimetrically from the most anterior part of the hippocampus to the caudal pole of this cortex. Area of the structures was measured at 28 magnification using a projector Documator DL-2 (Zeiss, Jena). The distance between serial sections used for planimetry was

0.4 mm. On average, 58 planimetric maps were used for control cases and 40 maps for AD cases. The area of the specific structure was multiplied by the distance between serial sections and the total volume of the structure was calculated.

Morphometry of Neurons. Tangles, Plaques, and Vessels With Amyloid Deposits Morphometry of neurons with and without neurofibrillary changes was performed along the whole rostrocaudal length of the hippocampal formation using six to eight sections 4 mm apart stained with mAb tau-1. Four stages of neurofibrillary changes were delineated based on the criteria proposed by Bancher et al. (7): stage 0, pretangle neurons that show scattered granular tau1-positive material in the cytoplasm; stage l, early stage tangles that are neurons with fibrillar and rod-shaped tau-l-positive deposits; stage 2, mature tangles that are neurons with cytoplasm partially or entirely filled with dense bundles of tau-l-positive material, and in which the shrunken nucleus is often dislocated; and stage 3, end-stage tangles that include both dense aggregates of tau-l-positive material, with no discernible nucleus, and clusters of extracellular filaments (ghost tangles), where immunostaining is variable and ranges from strong reactivity in large compact clusters to very weak or no reactivity in loosely arranged bundles of fibrils. The number of neurons stained with cresyl violet, and the number of tau-l-positive neurons and ghost tangles was determined at 560 magnification in the pyramidal layer of the cornu Ammonis, subiculurn proper, presubiculum, and parasubiculum, and in the granular layer of the dentate gyrus using a Pictoval projective microscope (Zeiss, Jena). Only neurons with visible nucleoli were counted. Test areas were positioned perpendicularly to the stratum oriens and radiatum in the cornu Ammonis or to the molecular layer in the subicular complex and included all layers (rows) of neurons in a given subdivision. In smaller subdivisions (CA2, CA3, CA4, presubiculum, and parasubiculum), test areas covered whole cross-sectional area (i.e., all neurons and NFTs on a given section were counted). In larger subdivisions (CA1 and the subiculum), test areas were randomly spaced and they covered the whole mediolateral extension of a given structure (in CA1, for example, test areas were systematically positioned from the border with CA2 to the oblique border with the subiculum). As noted above, the whole rostrocaudal length of the hippocampal formation was represented. The observed and computable variation of the estimate of the number of test areas for an individual was less than half that of the observed variance among individuals. The coefficient of error (CE) in this study was less than 0.045. The number of plaques and profiles of vessels stained with mAb 4G8 was measured using a projective microscope at magnification 165. Measurements were obtained in all layers of the cornu Ammonis, dentate gyrus, and subicular complex and in the whole thickness of the entorhinal cortex. For morphometry, a digitizer (Numonics) and a morphometric program (Sigma Scan-Jandel Scientific) were used. The numerical density and total number of neurons, tangles, and plaques were calculated according to equations (1) and (2), respectively (23,31).

NA Nv -At(t+ d - 2k)

(1)

where N v is the number of structures in a cubic millimeter; Na is the number of structures in the test area; A, is the test area (mm2); t is the section thickness (mm); d is the structure mean diameter (mm); and k is the correction factor. The correction factor k adjusts N v for those small cut caps of the structures that lie inside the section, yet cannot be visually discriminated. We calculated a k for

/

V

I

D

FIG. 1. Coronal, cresyl violet-stained sections of the hippocampal formation at the level of the hippocampal head (A,B), body (C,D), and tail (E,F) in control (B,D,F) and AD brain (A,C,E). Severe atrophy of all hippocampal formation subdivisions is present along the whole length of the AD hippocampus. Bar = 2 rr~aa. 911

912

BOBINSKI ET AL.

each subdivision in each case by focusing through the object and recording the microscope micrometer movement. In each AD and control case a random sample of neuronal nucleoli from six to eight sections representing the level of the hippocampal head, body, and tail were delineated using digitizer. The sample size was established with a 95% confidence interval (20). Long and short diameters were measured and a mean diameter (geometric diameter) of nucleolus of neuron with and without neurofibrillary changes was calculated for each examined hippocampal subdivision.

N t = VtNv

TABLE l VOLUME OF THE HIPPOCAMPAL FORMATION SUBDIVISIONS (ram3) Group Structure

Comu Ammonis Dentate gyms Subicular complex Entorhinal cortex

(2)

where N, is the total number of structures; and V~is the volume of the hippocampal subdivision. The shrinkage factor--relative measure of any distortion of the tissue that takes place during processing of the tissue blocks--did not vary among all slabs/sections for any case. There were no statistical differences in the shrinkage factors among all AD and control cases. No corrections for alterations of the tissue sample during histological processing were made in the present study. Statistical analysis was performed using the nonparametric Mann-Whitney U-test, Spearman's correlation rank test, and Pearson's correlation coefficient. RESULTS

Volume Volume of the hippocampal formation. In the control nondemented cases, the volume of the hippocampal formation was, on average, 3,009 mIn 3 (Table 1). In AD patients, the mean volume of the hippocampal formation was only 1,060 mm 3, a value 65% less than that in the nondemented, age-matched subjects (p < 0.05) (Fig. 1). The most severe difference was observed in the entorhinal cortex where the volume in the AD brains was 73% smaller than that of the controls (p < 0.05). The cornu Ammonis and subicular complex were affected very similarly; their volumes in the AD cases were 66 and 65% smaller (p < 0.05), respectively. The least difference was in the dentate gyms, which was only 38% smaller in AD (p < 0.05). Volume of sectors and layers of the cornu Ammonis. In AD, the most severe size differences were observed in sectors CA1 and CA2, with smaller volumes by 71 and 64% (p < 0.05), respectively. The volume of sector CA3 was smaller by 54% and of CA4 by 41% (p < 0.05). In AD brains, all layers of the cornu Ammonis were significantly smaller, but layers containing pyramidal neurons and their apical dendrites were most affected. The volumes of the strata pyramidale, radiatum, and lacunosurn/moleculare were smaller by 67, 77, and 65% (p < 0.05), respectively. The volumes of the alveus and stratum oriens were smaller by 52 and 57% (p < 0.05), respectively. Volume of layers of the dentate gyrus. In AD, the most severe size decrement was observed in the molecular layer, which was 49% less in volume (p < 0.05). Granular and polymorphic layers were relatively spared; their volumes were only 25 and 23% smaller (p < 0.05), respectively. Volume of parts and layers of the subicular complex. In AD, the greatest size difference in the subicular complex compared to controls was in the subiculum. Its volume was smaller by 70% (p < 0.05). The volumes of the presubiculum and parasubiculum were 57 and 56% smaller, respectively (p < 0.05). The most affected layer of the subicular complex was the pyramidal layer. Its volume was smaller by 71% (p < 0.05). The volumes of the parvopyramidal and molecular layers were 50 and 57% smaller (p < 0.05), respectively.

CA1 CA2 CA3 CA4 Alveus Stratum oriens Stratum pyramidale Stratum radiatum Stratum lacunosum/ moleculare Molecular layer Granular layer Polymorphic layer Subiculum Presubiculum Parasubiculum Molecular layer Pyramidal layer Parvopyramidal layer-presubiculum

Nondemented

AD

1,455 257 603 694

501 159 210 190

Sectors of the comu Ammonis 1,012 299 173 62 147 67 122 72 Layers of the comu Ammonis 169 81 119 51 654 216 230 54 282

99

Layers of the dentate gyrus 150 77 44 33 62 48 Parts of the subicular complex 375 112 176 75 52 23 Layers of the subicular complex 166 71 380 110 58

29

Neurons Numerical densi~ of neurons. In nondemented cases, numerical density of neurons in the pyramidal layer of the comu Ammonis ranged from 186/ram 2 in the CA4 sector to 420/ram 2 in the CA2 sector (Table 2). Numerical density of neurons in the pyramidal layer of the subicular complex showed regional differences and varied from 157/ram 2 in the subiculum to 212/ram 2 in the parasubiculum. The highest numerical density of neurons----4,110/ mm2--was seen in the granular layer of dentate gyms. AD cases showed significantly lower neuronal densities in all subdivisions of the hippocampal formation. As compared with nondemented brains, pyramidal cells, revealed by the examination of neuronal densities, in the immobile AD patients were 64% fewer in CA1, 31% fewer in CA2, 49% fewer in CA3, and 45% fewer in CA4 (p < 0.05). The density of neurons in the granular layer of the dentate gyrus was 29% less than controls (p < 0.05). In the subicular complex, significant neuronal loss based on neuronal density ranged from 37% in the presubiculum to 26% in the subiculum (p < 0.05). Total number of neurons and absolute neuronal loss. In nondemented cases, three-dimensional reconstruction revealed a total number of 8,639,000 neurons in the pyramidal layer of the cornu Ammonis; in the granular layer of the dentate gyms, 13,117,000; and in the pyramidal layer of the subicular complex, 3,671,000.

NEUROFIBRILLARY PATHOLOGY

913

TABLE 3 NUMERICAL DENSITY AND TOTAL NUMBER OF NEURONS WITH NFI's

'FABLE 2 NUMERICAL DENSITY .4ND TOTAL NUMBER OF NEURONS Numerical Density (N/mm2) Structure

Nondem,mted

AD

Comu Ammonis Subicular complex Dentate gyrus

--4,110

--2,917

Numerical Density (N/mm2)

Total Number (x 103) Nondemented 8,639 3,671 13,117

Total Number (×10 3)

AD

Structure

Nondemented

AD

Nondemented

AD

1392 825 7,474

Comu Ammonis Subicular complex Dentate gyrus

--16

--191

698 170 50

488 182 447

CA l CA2 CA3 CA4

228 420 351 186

Sectors of the comu Ammonis 82 5,896 289 728 180 807 103 1,208

493 236 241 422

CA1 CA2 CA3 CA4

22 56 6 3

Sectors of the cornu Ammonis 41 565 97 99 47 14 23 20

248 83 62 95

Subiculum Presubiculum Parasubiculum

157 165 212

Parts of the subicular complex 117 2,444 104 667 153 560

503 138 184

Subiculum Presubiculum Parasubiculum

11 2 1

Parts of the subicular complex 36 158 7 8 13 4

157 9 16

-Because of the significant differences among sectors of the comu Ammonis and parts of the subicular complex, average values for these subdivisions were not calculat,~.

-Because of the significant differences among sectors of the comu Ammonis and parts of the subicular complex, average values for these subdivisions were not calculated.

Total number of neurons irL the pyramidal layer was calculated to be 5,896,000 in the CA1 sector; 728,000 in the CA2 sector; 807,000 in the CA3 sector; and 1,208,000 in the CA4 sector. In the subicular complex, the largest total number of neurons was found in the subiculum (2,444,000), whereas the presubiculum and parasubiculum contained 667,000 and 560,000, respectively. The differences in neuronal numbers calculated from these total numbers of neurons were much higher than those based on numerical density of neurons in immobile AD patients. The absolute differences in neuronal numbers (AD vs. control) were 84% in the comu Ammonis, 78% in the subicular complex, and 43% in the dentate gyms (t7 < 0.05). In the cornu Ammonis, the greatest difference occurred in the CA1 sector with a difference in neuronal numbers of 92% (p < 0.05). In other sectors the results were similar: 68% in the CA2; 70% in the CA3; and 65% in the CA4 sector (p < 0.05). The subicular complex was also very severely affected. Neuronal numbers were 79% lower in AD both in the subiculum and presubiculum, and 67% in the parasubiculum (p < 0.05).

248,000 in the CA1 and 83,000 in the CA2 in AD cases. In sectors CA3 and CA4, the total number of neurons with NVl's was more than fourfold higher in AD cases than in the nondemented group. In the subicular complex, the total number of neurons with NFTs was similar in control (170,000) and AD cases (182,000). In the dentate gyrus, total number of neurons with NFTs was almost ninefold higher in AD cases (447,000) than in the nondemented group (50,000). Types of neurofibrillary tangles. The percentage of pretangles and early tangles (NFT 0 and NFT 1) was higher in nondemented than AD brain (Table 4). These types of NFTs constituted 63% of all tangles seen in the cornu Ammonis, 77% in the subicular complex, and 96% in the dentate gyrus in the nondemented group, and 41, 42, and 75% of all tangles, respectively, in the AD group. Mature and end-stage tangles (NFT 2 and N F r 3) prevailed in AD cases. In the cornu Ammonis, subicular complex, and dentate gyrus of the nondemented group, they constituted 37, 23, and 4% of all NFTs, respectively. In the AD group, the percentages of these types of NFTs were two- to sixfold higher (59, 58, and 25%, respectively).

Tangles Numerical density of neurons with neurofibrillary changes. Neurons with neurofibrillary changes were found in the pyramidal layer in all sectors of the cornu Ammonis in nondemented subjects. Their densities ranged from 3/mm 2 in the CA4 to 56/mm 2 in the CA2 (Table 3). There were 16/mm z neurons with neurofibrillary changes in the granular layer of the dentate gyms. In the pyramidal layer of the subicular complex, the densities of tangle-bearing cells ranged from l / m m 2 in the parasubiculum to 1 l / m m 2 in the subiculum. In the immobile AD cases, numerical densities of neurons with neurofibrillary degeneration were 2- to 13-times greater than in the control group.

Total numbers of neurons with neurofibrillary changes. The total number of neurons with NFTs in the cornu Ammonis in the immobile AD patients was 70% of the numbers in nondemented subjects (488,000 vs. 698,000). A greater total number of neurons with NFTs in nondemented cases than in AD group was shown only in sectors CAP and CA2. There were 565,000 neurons with NFTs in the CA1 and 99,0D0 in the CA2 in nondemented cases and

TABLE

4

STAGING OF NEUROFIBRILLARY TANGLES (%) Nondemented

Structure

NFr 0

NFF 1

17

46

26

32

45

71

25

AD

NVI" NFT 2 3

NFT 0

NFF 1

N V I ' NFT 2 3

11

15

26

37

22

16

7

12

30

40

18

4

0

44

31

17

8

ColTlU

Ammonis Subicular complex Dentate gyrus

Abbreviations: NVI" 0---pretangles; NFF 1---early tangles; NFT 2--mature tangles; NFF 3--end-stage tangles.

914

BOBINSKI ET AL.

Total number and percentage of neurons without neurofibrillary pathology. Whereas the total number of neurons in the pyramidal layer of the cornu Ammonis was 8.6 x 106 in the nondemented group, there were only 0.9 × 106 neurons without neurofibrillary tangles in the immobile AD patients (Table 5) (Fig. 2). In the subicular complex, these numbers were 3.7 × 10 6 and 0.6 × 106, respectively, and in the granular layer of the dentate gyrus 13.1 × 106 and 7.0 × 10 6, respectively. Neurons without neurofibrillary tangles constituted from 86% in CA2 to almost 100% in the granular layer of the dentate gyms in the nondemented group, and from 52% in CA1 to 94% in the granular layer of the dentate gyms in the AD group.

A

Plaques Numerical density of plaques in the hippocampal formation. Plaques were observed only in one case in the nondemented group and they were found only in the subicular complex (1.2/ram 2) and entorhinal cortex (0.9/ram2). AD cases showed rather uniform plaque densities through anatomical subdivisions of the hippocampal formation. The numerical density of plaques was 11.9/mm2in the cornu Ammonis, 14.5/mm2in the dentate gyms, 14.3/mm2in the subicular complex, and 16.l/ram 2 in the entorhinal cortex. In the cornu Ammonis, plaques were found in all layers (except the alveus), with the greatest density in the stratum radiatum (21.74/ mm2), fewer in the strata pyramidale ( 11.45/mm 2) and lacunosum/ moleculare (11.00/ram 2) and fewest in the stratum oriens (0.83/ mm2). In the subicular complex, numerical density of plaques was 24.92/mm 2 in the pyramidal layer and 1.43/ram 2 in the molecular layer. Total numbers of plaques in the hippocampal formation. In the one nondemented case, in which there were plaques, the reconstruction showed 13,500 plaques in the subicular complex and 2,700 in the entorhinal cortex. AD cases contained much larger numbers. The total number of plaques was 147,500 in the cornu Ammonis, 113,900 in the dentate gyms, 91,900 in the subicular complex, and 89,700 in the entorhinal cortex. Sector CA1 was much more affected than other sectors, with 115,400 plaques in CA1, 8,400 in CA2, 11,300 in CA3, and 12,400 in CA4. A predilection for plaque accumulation in specific layers was observed in the cornu Ammonis. The largest total number of plaques was in the stratum pyramidale (79,000), fewer in the stratum radiatum (37,000) and stratum lacunosum/moleculare (30,000), and only

B \

• []

CA1 [ ] CA2 • CA3 •

Subiculum Presublcutum Parasublculum

•

CA4 ~

Dentategyrus

FIG. 2. Diagram showing hippocampal formation subdivisions in nondemented control case (A) and immobile AD patient (B). Besides very prominent atrophy, illustrated in this figure, AD cases showed severe neuronal loss. The total numbers of neurons without neurofibrillary tangles in the immobile AD patient group were only 5% in CA1, 24% in CA2, 23% in CA3, 28% in CA4, 15% in subiculum, 20% in presubiculum, 30% in presubiculum, and 54% in the granular layer of the dentate gyrus of the total number of neurons without neurofibrillary tangles in nondemented group.

TABLE 5 TOTAL NUMBER (x 103) OF NEURONS WITHOUT NEUROFIBR1LLARY PATHOLOGY Structure

Nondemented

AD

Cornu Ammonis Subicular complex Dentate gyms

7,938 3,501 13,067

904 643 7,027

CAI CA2 CA3 CA4

Sectors of the cornu Ammonis 5,330 245 629 153 791 179 1,188 328

Subiculum Presubiculum Parasubiculum

Parts of the subicular complex 2,286 345 659 129 556 169

1,500 plaques in the stratum oriens. Plaques in the dentate gyms were found mostly in the molecular layer (107,300) and significantly less in the granular (4,800) and polymorphic layers (1,800).

Area of the layer occupied by amyloid deposits (amyloid burden). In the one nondemented case with plaques, amyloid deposits occupied 0.05% of the pyramidal layer in the subicular complex and 0.07% of the entorhinal cortex. In the cornu Ammonis of AD cases, plaques occupied from 0.73 % of the pyramidal layer of CA2 to 1.43% of the pyramidal layer of CA1. The granular layer of the dentate gyms was occupied in 1.16% by amyloid, and the pyramidal layer of the subiculum, presubiculum, and parasubiculum in 3.06, 2.14, and 3.32%, respectively. Amyloid angiopathy. No vessels with amyloid angiopathy were found in any control case. In AD cases, the numerical density of affected vessels in the hippocampal formation was similar and ranged from 0.42/mm 2 in the dentate gyrus to 1.8/mm 2 in the subicular complex. In the cornu Ammonis, affected vessels were

NEUROFIBRILLARY PATHOLOGY

915

observed in all layers (except alveus), but were most abundant in the pyramidal layer. The nuLmerical density of profiles of vessels with amyloid angiopathy was 1.32/ram 2 in CA1, 0.27/mm 2 in CA2, 1.83/mm 2 in CA3, aad 1.41/mm 2 in CA4. In the dentate gyrus, the density ranged from 0.25/ram 2 in the granular layer to 0.72/ram 2 in the molecular layer.

Statistical Analysis In the hippocampal formation subdivisions, the volumes and total numbers of neurons of each of the three nondemented control cases grouped in one cluster and those of the three AD cases grouped in the other cluster. The distance between the minimal value of the control group and the maximal value of the AD group always exceeded 2 SD (Fig. 3). Percentage change in volume was calculated for AD relative to the nondemented group (100%). Averaging the percentage change for the three AD cases, we examined the relationship between all hippocampal subdivision vol-

umes (pyramidal layer of CA1, CA2, CA3, CA4, subiculum, presubiculum, and parasubiculum, and granular layer of the dentate gyrus), and: (a) the percentage reduction in total numbers of neurons for the AD group relative to the nondemented group (r = ,83, p < 0.01); (b) percentage of neurons without neurofibrillary pathology among all neurons for the AD group (r = -.88, p < 0.001); (c) percentage of neurons with neurofibrillary pathology among all neurons for the AD group (r = .67, p < 0.05); (d) percentage of pretangles (stage 0) among all types of NFTs for the AD group (r = -.77, p < 0.01); (e) percentage of late stage tangles (stage 2) among all types of NFTs for the AD group (r = .78, p < 0.01). A linear correlation was found in the AD group between the percentage reduction in total number of neurons and the percentage of neurons with neurofibrillary pathology among all neurons for the AD group (r = .73, p < 0.05). There were no correlations between the percentage reductions in the volume of the hippocam-

CO'RNU A M M O N I S 1800

-F

1600

SUBICULAR COMPLEX 9 [xl06l

800

8

1400

7

1200

6

s [xlO'l

700 600

s N

mm31oool 80o 600 400

t

--;--

200 Volume

mm

3

4

4O0

32

300

1

200

0

100

Number of neurons

260 240

!

N

-3-

Volume

DF, N T A T E G Y R U S 280

3

500

N u m b e r of n e u r o n s

ENTORHINAL CORTEX 900

16 [xlO~]

800

T

14 700 12

220 mm 3

1o

60O

N

mm

3 500

2oo 40O 180

t

160 140 Volume

Number of neurons

300 200 4

-¢-

Max

Z Min o M e a n for control cases •

100

M e a n for A D cases

Volume

FIG. 3. Maximal, minimal, and mean values of volumes and total numbers of neurons for nondemented control cases (open squares) and AD cases (filled squares). The distance between minimal value of the control group and maximal value of the AD group always exceeded 2 SD. The total number of neurons for the entorhinal cortex was not estimated. The Duncan's multiple range test indicated no statistical differences among the three AD cases or among the three control cases, and, ther,efore, data are given as mean values for each group.

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BOBINSKI ET AL.

pal subdivisions in the AD group and each of the following parmeters: 1) the numerical density of plaques, 2) the total number of plaques, 3) the area of a layer occupied by amyloid, or 4) the numerical density of profiles of vessels with amyloid angiopathy. DISCUSSION Our previous volumetric study of 13 AD subjects (8) and this study, performed on a subset of three of these AD subjects, are in agreement in that both demonstrate that in AD the volumes of all subdivisions of the hippocampal formation are much smaller than in nondemented individuals. The present study differs from the previous study in that it describes neuronal loss and the total number, types, and distribution of NFrs and plaques in all subdivisions and layers of the hippocampal formation as well as hippocampal atrophy. The present study indicates that these volumetric losses were correlated with severe neurofibrillary changes and neuronal loss. The severe volume loss of the hippocampal formation in AD affects both cellular layers and layers composed of fibers to a similar extent. In the cornu Ammonis, volumetric loss from control values of the pyramidal layer (67%) was comparable to that in the stratum radiatum and lacunosum/moleculare (76 and 65%, respectively). The volumetric loss of the pyramidal layer reflects the loss of pyramidal cells, whereas the decrease in volume of the stratum radiatum and lacunosurrdmoleculare might be related to the loss of a) apical dendrites of the pyramidal cells and b) the perforant fibers and Schaffer collaterals. The 71% volumetric loss of the pyramidal layer in the subicular complex appears to be a direct effect of loss of cell bodies and perineuronal processes. The atrophy of the apical dendrites of pyramidal cells and of the dense network of fibers running through the molecular layer of the subicular complex, probably accounts for the 57% decrement in the volume of this layer. In immobile AD patients' brains, the volume of the granular and polymorphic layers of the dentate gyms decreased by 25 and 23%, respectively, in comparison with control volumes, while the atrophy of the molecular layer reached 49%. The granular layer is known to be mostly spared in the course of AD and is affected by neurofibrillary pathology only in late-stage AD (12). More severe volumetric loss in the molecular layer might be related to the formation of numerous [3-amyloid plaques, especially in the external two-thirds of this layer, i.e., in the area of the perforant pathway terminals (14,25,35). The differences in the magnitude of volumetric and neuronal loss among anatomically and functionally connected hippocampal structures suggests that the spread of atrophic changes might be not related to neuronal connections. The most severe atrophy was observed in the entorhinal cortex (73%), which is affected even in the preclinical stage of AD (8,12). Sectors CA1 to CA3 of the cornu Ammonis and subiculum, which receive direct projections through the alternative perforant path, belong to the most severely affected structures of the hippocampal formation. Their volumes decreased by 70, 64, 54, and 65%, respectively in comparison with control volumes. However, the dentate gyms, which is innervated by a main perforant pathway originating from the most early and most severely affected entorhinal cortex, revealed the latest onset of pathologic changes and the least atrophy (38%). Thus, the spread of pathologic changes in the hippocampal formation appears to be related to cell type-specific susceptibility to neurofibrillary pathology and structure- and layer-specific 13-amyloidaccumulation. Three-dimensional reconstruction of hippocampal formation subdivisions enabled analysis of the impact of neurofibrillary pathology, amyloid deposits, and amyloid angiopathy on the hippo-

campal subdivision volumes, and analysis of any correlations between atrophy and morphometric measurements of these pathologic lesions. Statistical analysis suggested that neurofibrillary pathology-not [3-amyloid deposition--underlies hippocampal volumetric loss. The relative decrease in the volume of the hippocampal subdivisions (pyramidal layer of sectors CA1-CA4, subiculum, pre and parasubiculum, and granular layer of the dentate gyms) correlated linearly with the relative decrease in the total number of neurons and neurons without NFTs. Furthermore, the relative decrease in the total number of neurons correlated with the relative increase in the total number of NFTs. However, the relative decrease in the volume of the hippocampal subdivisions was not correlated with numerical density or total number of plaques or the area occupied by amyloid or numerical density of profiles of vessels with amyloid angiopathy. The total number of neurons with and without NFTs found in the hippocampal formation in end-stage AD reflects the degree of morphological changes and functional decline of this part of the memory system. In the immobile AD patients, only 16% of the pyramidal cells survived in the cornu Armnonis, 22% in the subicular complex, and 57% of cells in the granular layer of the dentate gyms (Table 2). However, neurons without neurofibrillary pathology comprised only 11% in the comu Ammonis, 18% in the subicular complex, and 54% in the dentate gyms of those in nondemented cases (Table 5). The most severe decrease was observed in the CA1 and subiculum-neurons without NFTs comprised 5% and 15%, respectively, of those in the nondemented group. Interestingly, the total number of NFTs in some hippocampal subdivisions was greater in control cases than in AD. However, when the percentages they constitute in the total number of neurons are considered, these calculations show that despite their large total numbers in nondemented cases, Nk-Ts comprise only from 1.7% of neurons in CA3 and CA4 to 13% of neurons in CA2. In AD cases, tangle-bearing neurons constituted from 23% of the total number of neurons in CA4 to 50% of the total number of neurons in CA1. In the subicular complex, these percentages ranged from 0.7% in the parasubiculum to 7% in the subiculum proper in control cases, and from 9% in the presubiculum to 31% in the subiculum proper in AD cases. In the granular layer of the dentate gyms, which is regarded as virtually free of NFTs, tangles comprised 6% of the total number of neurons in the AD patient group and only 0.4% in the control subjects. The rate of maturation of NFTs is not known. The high percentages of mature and end-stage tangles in the cornu Ammonis (37%) and subiculum (23%) in the brains of nondemented subjects indicate that all steps of neurofibrillary degeneration, including disintegration of neurons, can occur before the onset of clinical symptoms. The low percentage (4%) of mature Nb-Ts and the lack of end-stage tangles in the dentate gyrus of the nondemented subjects, indicate a later onset of neurofibrillary pathology in this structure than in the comu Ammonis or subiculum. The greater relative number of mature (stage 2) and end-stage (stage 3) NFTs, i.e., twofold in the comu Ammonis and subicular complex and sixfold in the dentate gyrus in AD as compared with the nondemented group might mean that the process of maturation of NFTs is accelerated in AD. Our studies of neurofibrillary changes in aged schizophrenics treated with neuroleptics in comparison with untreated schizophrenics indicated that acceleration might also be induced by exogenous factors such as neuroleptics (58). If neurofibrillary changes can be promoted by some factors, perhaps other factors might inhibit or halt production of NFI's. The onset of neurofibriUary changes can be traced about 48

NEUROFIBRILLARY PATHOLOGY

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years before diagnosis of AD (44). Early onset of neurofibrillary degeneration may contribul:e to significant age-related neuronal loss in the transentorhinal cortex and hippocampal formation in young nondemented cases. The more pronounced neurofibrillary pathology in AD as compared to nondemented controls leads to severe neuronal loss. We fl)und that the total number of neurons was reduced in the immobile AD patient group in comparison with the control group by 84% in the pyramidal layer of the cornu Ammonis, by 78% in the l~yramidal layer of the subicular complex, and by 43% in the granular layer of the dentate gyrus. The most evident neuronal loss was observed in the pyramidal layer of the CA1 sector (92%); this subdivision was virtually devoid of neurons. In the other sectors of the cornu Ammonis, neuronal loss was very similar and ranged from 64% in CA2 to 70% in CA3. All parts of the subicular complex were also very severely affected. The total number of neurons in the pyramidal layer was decreased by 79% in both subiculum proper and presubiculum and by 67% in the parasubiculum. The magnitude of neuronal loss found in our study is more severe than that described l~y other authors. Ball (3) found a 47% loss of pyramidal neurons for the entire hippocampus. A significant reduction in the neuronal density in CA1 (HI sector) was reported to be 28% (21), 4J% (18), or 43% (43); in the subiculum, 39% (21), or 44% (18); artd in the prosubiculum, 28% (18). In a recent study, West et al. (56) found that the total number of neurons was significantly less than controls in the hilus (25%), CA1 (68%), and subiculum (47%) in AD cases; however, it was not significantly different frora controls in the granular layer of the dentate gyrus or in the CA3-CA2. We attribute the greater neuronal loss in our material to the use of brain material from very severe AD patients (GDS stage 7), who were averbal, nonambulatory, and immobile at the time of their demise, and who were assessed as having additionally lost the ability to sit up independently. Recent studies haw: demonstrated the continuing evolution of neurologic (24), cognitve (2), and physical (53) changes in these very severe stage 7 AD patients who have previously been grouped together as "end stage" in most studies. In accord with the advanced stage of AD in these patients, we noted a very long duration of disease (19-221 years from onset of clinically manifest subtle symptomatology in GDS stage 3 until demise). An additional reason for the magaitude of our findings may be our presentation of neuronal los:~ in terms of total number of neurons rather than neuronal density. Numerical density of neurons and cells with NFTs is a commonly used estimator of neuronal loss and neurofibrillary pathology (3,4,6,15,16,18,21,42,43). In the present study, when neuronal densities (N/mm 2) instead of total numbers of neurons were used, the intensity of neuronal loss was much less evident. Such calculations showed a decrease of 64% in CA1 (as

compared to 92% when calculated from total numbers); 31% in CA2 (64%), 49% in CA3 (70%), 45% in CA4 (65%), 29% in the dentate gyrus (43%), 26% in the subiculum (79%), 37% in the presubiculum (79%), and 28% in the parasubiculum (67%). Numerical density calculated per unit area or per unit volume does not take into consideration the actual volume of the structure, nor the volumetric changes in aging and dementia. Neuronal loss calculated from total numbers of neurons measures the cumulative effect of atrophy and decrement in numerical density. Because of a conflict between demands of diagnostic and immunocytochemical procedures used in this and other our studies, thin (8-p~m thick) sections and the Floderus method (23) for the estimation of the total number of neurons rather than the disector method (29,45, 46,54,57) using thick (at least 20-1xm thick) sections were applied in the present study. The role of amyloid 13 in AD is the subject of ongoing discussion (30,41,50). In our examined AD brains, the numerical density of plaques was rather uniform (from 11.9/mm 2 in the cornu Ammonis to 16.1/mm 2 in the entorhinal cortex). However, the impact of these amyloid deposits on the neuronal network of the hippocampal formation and on the progression of neurofibrillary pathology is not clear. Our data do not support a role for amyloid deposits and amyloid angiopathy in hippocampal atrophy, because no correlations were found between the relative decrease in the volume of the hippocampal subdivisions and each of the following parameters: 1) the numerical density of plaques, 2) the total number of plaques, 3) the area of a layer occupied by amyloid, or 4) the numerical density of vessels with amyloid angiopathy. The lack of plaques in two control cases and the low numerical density in the third one, as well as the presence of neurofibrillary changes in all nondemented cases (from 3/mm 2 in the CA4 to 56/mm 2 in the CA2) suggests that the onset of neurofibrillary pathology in the hippocampal formation is not related to [3-amyloid deposition. In subacute sclerosing panencephalitis (59) and in Guam dementia (32,33), neurofibrillary pathology develops and progresses without any 13-amyloid pathology. This and other studies indicate that the underlying processes associated with formation of plaques and tangles can be largely independent causatively (60), temporally (12), and spatially (52). ACKNOWLEDGEMENTS This work was supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and grants from the National Institutes of Health, National Institute on Aging No. AG 04220, AG 03051, AG 08051, and AG 12101 and the fund for the Center Trace Element Studies and Environmental Neurotoxicology. Dr. Kwang Sot Kim kindly supplied mAb 4G8; mAb tau-1 was the generous gift of Dr. Lester I. Binder.

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