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Regionally specific loss of neurons in the aging human hippocampus
Neurobiologyof Aging,Vol. 14, pp. 287-293, 1993
0197-4580/93$6.00 + .00 Copyright© 1993PergamonPress Ltd.
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Regionally Specific Loss of Neurons in the Aging Human Hippocampus MARK J. WEST
Stereological Research Laboratory University, Institute of Pathology and Second University Clinic of lnternal Medicine, Institute for Experimental Clinical Research and Institute for Neurobiology, University of Aarhus, Denmark Received 10 August 1992; Revised 3 February 1993; Accepted 10 February 1993 WEST, M. J. Regionallyspecific loss of neurons in the aging humanhippocampus. NEUROBIOLAGING 14(4) 287-293, 1993.New highly-accuratestereologicalmethods for estimatingthe total numbersof neuronsin brain structures have been used to test for age-related neuron loss in the human hippocampalformation. Across the age range of 13 to 85 years, there was a substantialloss of neuronsin the subiculum(52%) and in the hilus of the dentate gyrus (31%); the three remaininghippocampalsubdivisionsshowed no significantchange. These losses qualify as potentialmorphologicalcorrelates of senescentdecline in relationalmemory in that they can be expected to compromisethe functionalintegrityof a regionof the brainknownto be intimatelyinvolvedin this type of memory. The regional pattern of neuron loss is similar in certain respects to the patterns of cell loss seen during the initial phases of ischemia and epilepsy and is fundamentallydifferent from the pattern associated with Alzheimer's disease. Aging
Hippocampus
Neuronl o s s
Memory
Stereology
NORMAL human aging is generally accompanied by a progressive decline in performance on memory tasks involving new information about configurations and relationships (32,33). The identification of morphological correlates of this decline can contribute to a better understanding of senescent memory dysfunction and aid in the identification of the mechanisms involved in the apparent selectivity of senescent memory deficits. Several previous studies have focused on age-related neuron loss in the human hippocampal formation because of its known role in relational memories (5,27,30). For methodological reasons, these studies have led to uncertainties about the extent and even the existence of age-related neuron loss in the human hippocampal formation (10). In the present study, age-related neuron loss in the human hippocampal formation has been re-examined using recently developed stereological methods, (i.e., methods for the quantitative study of three-dimensional structure with geometrical probes) that eliminate virtually all of the deficiencies of previously available techniques (42). METHOD The material used in this study was obtained from autopsies performed at Danish hospitals. It comprised the left temporal lobes of 32 males, who ranged from 13 to 85 years of age and had no histories of neurological or psychiatric disorders, prolonged illness, metastatic cancer, drug, or alcohol abuse. The post-mortem delay prior to fixation ranged from 6 to 48 h. The entire brains were fixed in 4% phosphate buffered formalin for 7 to 72 months.
Disector
detail previously (43). Salient features of these include the following: The analysis was performed on 70 Ixm thick glycolmethacrylate sections cut in the frontal plane and sampled at 3.5 mm intervals along the entire anterior-posterior extent of the left hippocampal formation. The neuron containing layers of five major subdivisions of the hippocampal formation were outlined on each of the 10 to 14 slides used from each individual. These included (a) the granule cell layer, (b) the hilus of the of the dentate gyrus, (c) CA3-2, (d) CA1, and (e) the subiculum (Fig. 1 A,B). The exact boundaries of the subdivisions of the hippocampal formation, as defined and used in this study, have been described in detail previously (43). Notable aspects of the definitions of the subdivisions used here are the inclusion of the prosubiculum in CA1 and the inclusion of the CA4 pyramidal ceils (35) or the "end blade" of CA3 (1) in the hilus, in accordance with the descriptions of Geneser (14). The decisions about the boundaries to be used were dictated as much by practical considerations as by functional considerations in that the methodology requires consistent, well defined boundaries along the entire extent of the hippocampal formation. An unbiased estimate of the total number of neurons (N) in each subdivision of each individual was derived from the product of an unbiased estimate of the numerical density of neuronal nuclei, i.e., number per unit volume (Nv) , and an unbiased estimate of the volume of the regions of the subdivisions that contained neuronal cell bodies (Vref) according to the following equation.
N = Nvo Vr,y
[EQ. 1]
The N v was estimated in each individual subdivision from the counts obtained from approximately 100 optical disectors (16,17, 42,43) made with a Leica Stereological Analysis Microscope System. The optical disectors used in the analysis were dimensioned
Stereological Analysis The preparation of the tissue used in the stereological analysis and the stereological methods themselves have been described in 287
288
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h
B
Gra%
~
Hilus
CA3 -2
FIG. 1. Structural organization of the human hippocampal formation. (A) Histological section through the dorsal-medial part of the temporal lobe of C the brain showing the transverse organization of the architectonic subdivisions of the hippocampal formation. Medial to the left. Micrograph width = 2 cm. (B) Delineation of the subdivisions of the hippocampal formation in which estimates of the total numbers of neurons were made. Gran, the granule cell layer of the dentate gyrus. Hilus, the hilus of the dentate gyrus Gran ~ 2 which contains both neurons of the polymorphic layer and the pyramidal I neurons of the inner cellular layer (14). CA3-2, the layer containing tightly packed cell bodies of the large pyramidal neurons of the hippocampus proper. CA1, the layer containing the cell bodies of smaller, radially dispersed pyramidal neurons of the hippocampus proper. Sub, the subiculum, a thick zone of pyramidal neurons organized in two layers. The dotted subdivisions, hilus and sub, are those in which there were significant age-related losses of neurons. Estimates were not made in entorhinal cortex, EC, which has been illustrated for the sake of the discussion of the .l . . . . L I results. (C) Diagrammatic representation of the major connections of the sub1 i divisions. Axons of the granule cells (1) synapse on the polymorphic EC J' i I neurons of the Hilus (a) and pyramidal neurons of the Hilus (b) and CA3. i The polymorphic neurons of the Hilus (a) provide both excitatory and inhibitory feedback connections to the dentate granule cells (2). The pyramidal neurons of the Hilus (b), like those of CA3 (see ref. 1, p. 723) project to CA1 (3). The pyramidal neurons of CA2, which were included in the same zone as the pyramidal neurons of CA3, similarly project to Cortex CAI but unlike the pyramidal cells of CA3, do not receive input from the granule cells. The pyramidal neurons of CAI project to the subiculum (4), which in turn, projects to parahippocampal cortical regions, including the entorhinal cortex (5). The entorhinal cortex, in addition to having reciprocal connections with a wide range of cortical association areas, is the major source of extrinsic afferents to the dentate granule cells and pyramidal cells of the hippocampus proper. The age-related losses of neurons in the hilus and the subiculum (shaded regions) compromise the processing of information within the hippocampal formation and the transmission of hippocampal information to other cortical areas.
T-
so that 1-2 neurons, on average, were counted in each disector sample. Unbiased estimates of Vre/ were obtained with Cavalieri's point counting method (18,26) using a total of approximately 100 points in each subdivision. The sampling with both disectors and points was carried out systematic random in all three dimensions of each subdivision to ensure that all parts of the subdivision had an equal probability of being sampled. The first section was selected from a random position within the first 3.5 m m interval and the following sections selected at regular 3.5 m m intervals thereafter. On each section, the disectors and points were placed randomly within the first x,y interval of the areal profile of a region and at regular intervals in a raster pattern thereafter. The amount of sampling, i.e., the intervals between samples, was set a priori on the basis of a preliminary analysis of the inter-individual variance and the precision of the individual estimates made in a small number of individuals (43). At the end of the study it was confirmed that the major factor contributing to the
observed inter-individual variability was the fixed, inherent, biological variability of the individuals, and not the precision of the individual estimates. In practice this was accomplished by ensuring that the observed relative variance of the individual estimates, CE 2, was less than the fixed, inherent relative variance of the group, ICV 2, and thereby made a smaller contribution to the observed relative variance of the group, C V 2, according to the following relationship. (See ref. 42 for additional details.) CV 2 = ICV 2 + CE 2
IEQ. 21
For subdivisions in which there eventually were shown to be correlations between number and age, the C V 2 used in this analysis was that of the residual variance, i.e., that not explained by age. Staining f o r Senile Plaques and Neurofibrillary Tangles Single sections, taken from the midpoint along the rostralcaudal axis of the hippocampi from the each of the 17 subjects
REGIONALLY SPECIFIC LOSS OF NEURONS
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FIG. 2. The total number of neurons (millions) in each of the five subdivisions of the hippocampal formation plotted as a function of age (years). The regression line for number versus age is shown in the two subdivisions in which there were statistically significant correlations between neuron number and age.
older than 50 years of age, were stained for senile plaques (SP) and neurofibrillary tangles (NFT) with thioflavin-S (4). A modification of the semiquantitative scale used by Kromer-Vogt et al. (21) was used to assess the presence of SPs and NFTs in six subdivisions of the hippocampal formation and in temporal neocortex. RESULTS
Neuron Numbers The total number of neurons in each subdivision of each of the 32 individuals is shown graphically in Fig. 2 as a function of age. There were statistically significant negative correlations between neuron number and age in the hilus and subiculum (Table 1), representing 31% and 52% reductions, respectively, in the number of neurons in these two zones over the ages studied. In terms of the reduction in the number of neurons per decade, this translates to 89,000 or 4.2% of the neurons in the hilus and 550,000 or 7.3% of the neurons in the subiculum. There were no statistically significant correlations between age and the numbers of neurons in the dentate granule cell layer and the pyramidal cell layers of C A 3 - 2 and CA1. The most noteworthy aspect of the age-related loss of neurons is that it is confined to two of the five subdivisions of the hippocampal formation, hilus and subiculum. The estimates
of the total number of neurons in each of the subdivisions of each of the individuals included in this study are shown in Table 2.
Evaluation of Plaques and Tangles In 13 of the 17 subjects that were over the age of 50 years, there was evidence of either SP or neurofibrillary tangles NFT in the hippocampal and parahippocampal regions. The numbers of SPs and NFTs per m m 2 of the most affected parts of the six hippocampal subdivisions and, when available, temporal neocortex are shown in Table 3. On the basis of the CERAD criteria for the neuropathological diagnosis of Alzheimer's disease (AD) (28), one of the subjects was classified as a " p o s s i b l e " case of AD (Tables 2 and 3; Subject 29). The removal of this individual from the study did not affect the conclusions drawn with regard to the presence of statistically significant age-related losses of neurons in the hilus (r = - 0 . 3 9 5 , 2 p = 0.028) and the subiculum (r = - 0 . 4 3 9 , 2 p = 0.013). DISCUSSION
Age-Related Neuron Loss The data presented here provide the first evidence for an agerelated reduction in the total number of neurons in a part of the
TABLE 1 STATISTICAL PARAMETERS CHARACTERIZINGRELATIONSHIP BETWEEN TOTAL NEURON NUMBER AND AGE
Gran Hilus CA3-2 CA1 Subic
r
2p
- 0.11 -0.38 -0.21 -0.11 -0.46
0.55 0.032 0.24 0.53 0.008
b
-0.009
-0.035
N
CV
19.4 1.78 2.82 14.4 6.16
0.28 0.31 0.23 0.26 0.27
UCV
% Lost
0.29
31%
0.24
52%
At the end of the analysis it was confirmed, for each subdivision, that the major factor contributing to the observed relative variance of the group, CV 2, was the fixed, inherent variance of the group, ICV 2, and not the variance of the individual estimates, CE 2, which is related to the precision of the stereological procedures and the amount of sampling (42). In subdivisions in which there were eventually shown to be correlations between number and age, the residual variance, UCV 2, i.e., that not explained by age, was used instead of the CV 2. In the five subdivisions, the fixed, inherent variance of the group, 1CV 2, comprised 85%, 70%, 68%, 84%, and 85%, respectively, of the observed relative variance of the group (see Eq. 2). r = Pearson product moment correlation coefficient; 2p = significance level; b = regression coefficient, millions of neurons per year; N = mean total number of neurons in millions (n = 32); CV = coefficient of variation, (SD)/(mean); UCV = relative variation of total number, N (n = 32), (SD)/(mean), unexplained by age; % lost = based on regression value at age 13 years.
290
WEST
TABLE 2 TOTAL NUMBER OF NEURONS (MILLIONS) IN EACH OF THE SUBDIVISIONS OF THE HIPPOCAMPAL FORMATION OF 32 SUBJECTS OF VARYING AGE Subject
Age
Gran
Hilus
CA3 2
CAI
Sub
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
13 15 19 21 21 21 28 34 38 41 42 43 43 44 50 52 56 56 58 58 63 64 66 68 73 78 78 78 80 80 84 85
19.95 16.69 19.38 23.98 11.21 15.02 29.08 14.29 25.48 27.88 19.55 16.18 31.40 20.17 18.50 12.75 15.51 10.88 18.12 21.03 30.57 24.80 17.61 21.27 18.44 20.47 18.44 15.75 22.26 12.20 11.77 19.26
3.24 1.82 2.18 1.86 1.70 2.78 1.66 1.59 1.33 2.35 1.63 1.42 1.60 1.98 2.39 1.16 1.68 1.71 1.93 2.02 2.58 1.54 1.17 0.81 1.36 0.74 1.29 1.47 1.83 1.84 1.67 2.51
4.07 2.52 3.55 2.00 3.07 2.36 3.32 2.62 2.66 2.08 2.23 4.21 2.67 3.33 2.83 2.95 3.64 3.00 2.25 3.32 3.75 3.14 2.25 2.13 2.64 1.45 3.37 2.44 2.53 2.00 2.46 3.44
19.05 11.83 15.50 13.67 9.91 22.88 15.57 10.53 12.64 11.22 19.80 13.79 16.85 11.77 12.84 7.37 9.38 15.56 21.04 20.96 17.17 16.13 12.59 10.18 17.61 12.38 I 1.77 15.13 14.58 I 1.84 16.11 11.42
5.97 5.61 7.44 6.81 7.93 6.80 10.19 7.52 7.83 3.54 6.48 5.97 6.71 8.36 7.64 4.59 5.73 5.46 6.84 4.67 5.56 8.08 6.36 3.87 7.99 2.89 5.23 6.84 4.54 4.68 5.83 3.08
formation o f y o u n g e r generations, rather than n e u r o n loss. T h e r e is, at present, no w a y to formally d i s c o u n t this interpretation, in that it w o u l d require the analysis o f material collected over the past or next 72 years. O n e reason for rejecting this interpretation, however, is that the correlation b e t w e e n age and n e u r o n n u m b e r was observed in only two o f the subdivisions. Secular c h a n g e s , like intraspecific differences, are m o s t c o m m o n l y associated with differences in the overall size o f individuals and w o u l d be expected to be a c c o m p a n i e d by geometrical scaling o f brain c o m p o n e n t s , i.e., the parts o f the brain would scale in constant proportion to each other (15).
The Presence of AD T h e presence o f plaques and tangles in the h i p p o c a m p u s and temporal cortex o f a majority o f the older subjects s u g g e s t s that A D m a y be responsible the age-related losses reported here. There are, h o w e v e r , several r e a s o n s for not adopting a strict stance with regard to the elimination o f individuals with e v i d e n c e o f plaques TABLE 3 EVALUATION OF NEUROFIBRILLARY TANGLES (NFT) AND SENILE PLAQUES (SP) Subject 16 17 18 19 20 21 22 23
h u m a n brain k n o w n to be involved in m e m o r y processes that decline with age. T h e loss o f n e u r o n s in the hilus and s u b i c u l u m thereby qualify as potential m o r p h o l o g i c a l correlates o f s e n e s c e n t decline in relational m e m o r y . A l t h o u g h o n g o i n g studies d e s i g n e d to analyze the covariation b e t w e e n n e u r o n n u m b e r and perform a n c e on relational m e m o r y tasks m u s t be c o m p l e t e d before drawing definitive c o n c l u s i o n s about the relationship b e t w e e n these two p h e n o m e n a , several points c a n be m a d e to e m p h a s i z e the functional implications o f these losses and the perspectives that the regional selectivity o f the losses h a v e for the identification o f the m e c h a n i s m s involved. Prior to doing so, h o w e v e r , it is appropriate to discuss w h y secular c h a n g e s and the presence o f A D are improbable explanations o f the age-related reductions in neuron n u m ber o b s e r v e d here.
24 25 26 27 28 29 30 31
Secular Changes B e c a u s e the material u s e d in this s t u d y is a cross-sectional s a m p l e o f the entire a g e r a n g e that w a s collected over a 3-year period, it is possible that secular c h a n g e s rather than selective, regional, age-related, n e u r o n losses are responsible for the agerelated differences in the n u m b e r s o f n e u r o n s in the hilus and s u b i c u l u m . T h a t is, the differences could reflect a consistent trend toward m o r e n e u r o n s in specific s u b d i v i s i o n s o f the h i p p o c a m p a l
32
PHG NIT SP NFT SP NFT SP NFT SP NFT SP NFT SP NFT SP NIT SP NFf SP NFI" SP NFT SP NFf SP NFT SP NFT SP NFT SP NF'r SP NFT SP
0 0 0 0 + ++ + 0 +* 0 0 0 0* 0 + 0 + 0 +* +-+ + + + +* ++ + +* 0 + + +* ++ 0 0 +* 0 + + +
DG Hil CA3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CAI
Sub
0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + 0 + + + + + 0 0 0 + +
0 0 0 0 0 ++ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + 0 + + + ++ + + + + + 0 +
Ctx 0 0 0 0 0 0 0 -+ + 0 +++ + + +++ 0 0 -
PHG = parahippocampal gyms; DG = dentate gyms; Hil = dentate hilus; CA3 = CA3; CA1 = CA1; Sub = subiculum; Ctx = temporal neucortex; 0 = 0 NFT or SP/mm2; + = 1-5 NFT or SP/mm2; + + = 6--10NFTorSP/mm2; + + + = 11-25NFrorSP/mm2; ++++ = >25 NF'I" or SP/mm2; * = entorhinal cortex; - = no observations.
REGIONALLY SPECIFIC LOSS OF NEURONS and tangles from this study of normal aging. First and foremost, the relationship between normal aging processes and the processes involved in the formation of plaques and tangles is not fully understood. Both plaques and tangles are observed in nondemented individuals (4,40) and are not unequivocal evidence of AD. The neuropathological criteria recommended by NINCDS-ADRDA and CERAD, for a neuropathological diagnosis of AD, both involve age-adjusted plaque counts (20,28), indicating that, by definition, the mere presence of plaques is not adequate for the diagnosis. Although the criteria themselves are the subject of debate (13), primarily as a consequence of the difficulties encountered when establishing threshold values for a neuropathological diagnosis and the poor correlation between the severity dementia and number of SPs (3), it could be argued that the one possible case (Tables 2 and 3; Subject 29) should be excluded from this study on the basis of neuropathological criteria. The removal of this case, however, does not alter the main conclusion drawn from this study, i.e., that there are statistically significant age-related losses of neurons in the hilus and the subiculum. There are two reasons for not excluding any of the subjects from this study on the basis of suspected AD. First, the clinical criteria, which are used in concert with the neuropathological criteria when diagnosing AD according to the CERAD and NINCDS-ADRDA guidelines, are not fully satisfied by any of the subjects used in this study. Although they were not explicitly tested psychometrically for signs of dementia, there was no evidence in their medical records of neurological or psychiatric disorders. All died under relatively acute circumstances involving little or no hospitalization and apparently conducted normal lives prior to their deaths. Second, the pattern of neuron loss reported here is qualitatively different from that observed in advanced cases of AD. Preliminary evaluations of the regional neuron loss in advanced cases of AD indicates that it is the CA1 region, and not the hilus or the subiculum, that selectively looses neurons in AD patients that meet both clinical and neuropathological criteria (44). The presence of SPs and NFFs in 13 of the 17 of the subjects over the age of 50 years does underline both the practical problems involved in obtaining aged subjects who are free of the pathological hallmarks of AD and the uncertainty surrounding the concept of normal, pathology free, aging. The final resolution of the issue as to when individuals with plaques and tangles should be excluded from a study of normal aging awaits a more complete understanding of the relationship between normal aging processes and the processes involved in the formation of plaques and tangles. With this caveat in mind, along with that with regard to the secular interpretation of the data described above, the data will be discussed exclusively in terms of neuron loss related to normal aging.
Functional Implications of the Neuron Loss In view of what is known about the functional organization of hippocampal neurons, the reported losses can be expected to reduce the information processing capacity of the hippocampal formarion. The hippocampal subdivisions are connected by a unidirectional system of parallel and serial neuronal circuits through which information flows from the dentate gyms to the subiculum (2) (Fig. 1C). The neurons in the hilus participate in the flow of information though the initial series of circuits in the hippocampal formation. A reduction in the number of these neurons would be expected to reduce the associative capacity of the dentate granule cells and the amount or resolution of the information sent from this region to CA1 (7,23,31). The loss in subiculum may represent a more profound deficit because of the relative magnitude of the neuron loss and its position at the end of the unidirectional system
291 of fiber connections within the hippocampal formation. There it serves as the major distributor of hippocampal output information to other cortical regions, including entorhinal cortex (45), that are intimately involved in the "back projections" required to establish long term memories (34,39). Although it has been suggested that age-related neuron loss can be viewed as a continuation of normal developmental processes that fine tunes neuronal circuitry and may even have functional benefits (24), there are limits to the degree to which a reduction in the number of neurons within specific subdivisions of the hippocampal formation can be construed as beneficial. Disruptions in the flow of information through the hippocampal formation resulting from regional neuron loss produce anteriograde amnesia in humans (46) and deficits in performance on specific relational memory tasks in experimental animals (29,47). A determination of the point at which neuron loss translates to functional impairment must await the results of ongoing studies in humans and nonhuman primates designed to evaluate the covariation between the number of neurons in specific hippocampal subdivisions and performance on relational memory tests. The results of these studies will also be helpful in determining the degrees to which compensatory physiological, structural, and biochemical processes, both inside and outside the hippocampal formation are involved in senescent decline in relational memories.
Regional Selectivity The regional selectivity of the age-related neuron loss reported here may provide clues to the mechanisms involved in the loss and maintenance of specific groups of neurons during aging. Promising directions for future research include more detailed examinations of the relationship between neuron loss and Alz-50 immunoreactivity (8), acetlycholinesterase-rich neurons that develop later in life (25), and the regional distribution of receptor systems that mediate excitatory synaptic transmission (11). The latter is of particular interest because specific subgroups of these receptors have been shown to be involved in excitotoxic neuron death that is associated with a number of pathological states and can be potentiated by stress and glucocorticosteroids (36). The age-related loss of neurons observed in the hilus warrants additional study to determine whether the specific subgroups of hilar neurons, reported to be involved in the initial pathological changes related to ischemia (19), epilepsy (12,37,38), and those associated with AD (9) contribute disproportionately to the observed loss and provide evidence of the participation of neurodegenerative mechanisms related to these other phenomena. The regional pattern of age-related neuron loss is similar but not identical to that seen in the hippocampal formation of patients with AD. Qualitative and quantitative studies clearly indicate that the CA1 region is one of the most heavily afflicted regions in AD patients (6,41) and undergoes extensive neuron loss in advanced cases (44). In spite of similarities in the losses of neurons in the hilus and the subiculum of normal-aged subjects and AD patients, the lack of evidence of an age-related change in the number of CA1 neurons, in the data presented here, strongly suggests that there is a fundamental qualitative difference between the regional pattern of neuron loss associated with normal aging and that associated with AD and a fundamental difference in the mechanisms (22) involved in the neuron loss associated with the two processes. ACKNOWLEDGEMENTS I thank Dr. Bradley Hyman (Neurology Service, Massachusetts General Hospital, Harvard Medical School, Boston, MA) for his assistance with the staining and evaluation of the thioflavine-S preparations. Also, I thank Maj-Britt Lundorf for assistance with the histology; Anette Larsen
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and Albert Meier for assistance with the illustrations; the Departments of Pathology at Aarhus Kommunehospitalet and Aarhus Amtssyghus, the Department of Forensic Medicine, Aarhus University, and The Neurological Research Laboratory, Bartholin Institute, Copenhagen for providing the histological material used in this study; Henrik Clausen for his roll in the development of the GRID stereological software package; Erik MOiler
of the Danish office of Leica, Glostrup, Denmark for assistance with the microscope equipment used for the stereological analysis. This work has been supported by The Danish Health Sciences Research Council, Aarhus University Research Foundation, Fonden til Forskning af Sindslidelser, Fonden af 1870, and the Danish Foundation for Experimental Research in Neurology.
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REGIONALLY SPECIFIC LOSS OF NEURONS
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0197-4580/93$6.00 + .00 Copyright© 1993PergamonPress Ltd.
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Regionally Specific Loss of Neurons in the Aging Human Hippocampus MARK J. WEST
Stereological Research Laboratory University, Institute of Pathology and Second University Clinic of lnternal Medicine, Institute for Experimental Clinical Research and Institute for Neurobiology, University of Aarhus, Denmark Received 10 August 1992; Revised 3 February 1993; Accepted 10 February 1993 WEST, M. J. Regionallyspecific loss of neurons in the aging humanhippocampus. NEUROBIOLAGING 14(4) 287-293, 1993.New highly-accuratestereologicalmethods for estimatingthe total numbersof neuronsin brain structures have been used to test for age-related neuron loss in the human hippocampalformation. Across the age range of 13 to 85 years, there was a substantialloss of neuronsin the subiculum(52%) and in the hilus of the dentate gyrus (31%); the three remaininghippocampalsubdivisionsshowed no significantchange. These losses qualify as potentialmorphologicalcorrelates of senescentdecline in relationalmemory in that they can be expected to compromisethe functionalintegrityof a regionof the brainknownto be intimatelyinvolvedin this type of memory. The regional pattern of neuron loss is similar in certain respects to the patterns of cell loss seen during the initial phases of ischemia and epilepsy and is fundamentallydifferent from the pattern associated with Alzheimer's disease. Aging
Hippocampus
Neuronl o s s
Memory
Stereology
NORMAL human aging is generally accompanied by a progressive decline in performance on memory tasks involving new information about configurations and relationships (32,33). The identification of morphological correlates of this decline can contribute to a better understanding of senescent memory dysfunction and aid in the identification of the mechanisms involved in the apparent selectivity of senescent memory deficits. Several previous studies have focused on age-related neuron loss in the human hippocampal formation because of its known role in relational memories (5,27,30). For methodological reasons, these studies have led to uncertainties about the extent and even the existence of age-related neuron loss in the human hippocampal formation (10). In the present study, age-related neuron loss in the human hippocampal formation has been re-examined using recently developed stereological methods, (i.e., methods for the quantitative study of three-dimensional structure with geometrical probes) that eliminate virtually all of the deficiencies of previously available techniques (42). METHOD The material used in this study was obtained from autopsies performed at Danish hospitals. It comprised the left temporal lobes of 32 males, who ranged from 13 to 85 years of age and had no histories of neurological or psychiatric disorders, prolonged illness, metastatic cancer, drug, or alcohol abuse. The post-mortem delay prior to fixation ranged from 6 to 48 h. The entire brains were fixed in 4% phosphate buffered formalin for 7 to 72 months.
Disector
detail previously (43). Salient features of these include the following: The analysis was performed on 70 Ixm thick glycolmethacrylate sections cut in the frontal plane and sampled at 3.5 mm intervals along the entire anterior-posterior extent of the left hippocampal formation. The neuron containing layers of five major subdivisions of the hippocampal formation were outlined on each of the 10 to 14 slides used from each individual. These included (a) the granule cell layer, (b) the hilus of the of the dentate gyrus, (c) CA3-2, (d) CA1, and (e) the subiculum (Fig. 1 A,B). The exact boundaries of the subdivisions of the hippocampal formation, as defined and used in this study, have been described in detail previously (43). Notable aspects of the definitions of the subdivisions used here are the inclusion of the prosubiculum in CA1 and the inclusion of the CA4 pyramidal ceils (35) or the "end blade" of CA3 (1) in the hilus, in accordance with the descriptions of Geneser (14). The decisions about the boundaries to be used were dictated as much by practical considerations as by functional considerations in that the methodology requires consistent, well defined boundaries along the entire extent of the hippocampal formation. An unbiased estimate of the total number of neurons (N) in each subdivision of each individual was derived from the product of an unbiased estimate of the numerical density of neuronal nuclei, i.e., number per unit volume (Nv) , and an unbiased estimate of the volume of the regions of the subdivisions that contained neuronal cell bodies (Vref) according to the following equation.
N = Nvo Vr,y
[EQ. 1]
The N v was estimated in each individual subdivision from the counts obtained from approximately 100 optical disectors (16,17, 42,43) made with a Leica Stereological Analysis Microscope System. The optical disectors used in the analysis were dimensioned
Stereological Analysis The preparation of the tissue used in the stereological analysis and the stereological methods themselves have been described in 287
288
WESq'
h
B
Gra%
~
Hilus
CA3 -2
FIG. 1. Structural organization of the human hippocampal formation. (A) Histological section through the dorsal-medial part of the temporal lobe of C the brain showing the transverse organization of the architectonic subdivisions of the hippocampal formation. Medial to the left. Micrograph width = 2 cm. (B) Delineation of the subdivisions of the hippocampal formation in which estimates of the total numbers of neurons were made. Gran, the granule cell layer of the dentate gyrus. Hilus, the hilus of the dentate gyrus Gran ~ 2 which contains both neurons of the polymorphic layer and the pyramidal I neurons of the inner cellular layer (14). CA3-2, the layer containing tightly packed cell bodies of the large pyramidal neurons of the hippocampus proper. CA1, the layer containing the cell bodies of smaller, radially dispersed pyramidal neurons of the hippocampus proper. Sub, the subiculum, a thick zone of pyramidal neurons organized in two layers. The dotted subdivisions, hilus and sub, are those in which there were significant age-related losses of neurons. Estimates were not made in entorhinal cortex, EC, which has been illustrated for the sake of the discussion of the .l . . . . L I results. (C) Diagrammatic representation of the major connections of the sub1 i divisions. Axons of the granule cells (1) synapse on the polymorphic EC J' i I neurons of the Hilus (a) and pyramidal neurons of the Hilus (b) and CA3. i The polymorphic neurons of the Hilus (a) provide both excitatory and inhibitory feedback connections to the dentate granule cells (2). The pyramidal neurons of the Hilus (b), like those of CA3 (see ref. 1, p. 723) project to CA1 (3). The pyramidal neurons of CA2, which were included in the same zone as the pyramidal neurons of CA3, similarly project to Cortex CAI but unlike the pyramidal cells of CA3, do not receive input from the granule cells. The pyramidal neurons of CAI project to the subiculum (4), which in turn, projects to parahippocampal cortical regions, including the entorhinal cortex (5). The entorhinal cortex, in addition to having reciprocal connections with a wide range of cortical association areas, is the major source of extrinsic afferents to the dentate granule cells and pyramidal cells of the hippocampus proper. The age-related losses of neurons in the hilus and the subiculum (shaded regions) compromise the processing of information within the hippocampal formation and the transmission of hippocampal information to other cortical areas.
T-
so that 1-2 neurons, on average, were counted in each disector sample. Unbiased estimates of Vre/ were obtained with Cavalieri's point counting method (18,26) using a total of approximately 100 points in each subdivision. The sampling with both disectors and points was carried out systematic random in all three dimensions of each subdivision to ensure that all parts of the subdivision had an equal probability of being sampled. The first section was selected from a random position within the first 3.5 m m interval and the following sections selected at regular 3.5 m m intervals thereafter. On each section, the disectors and points were placed randomly within the first x,y interval of the areal profile of a region and at regular intervals in a raster pattern thereafter. The amount of sampling, i.e., the intervals between samples, was set a priori on the basis of a preliminary analysis of the inter-individual variance and the precision of the individual estimates made in a small number of individuals (43). At the end of the study it was confirmed that the major factor contributing to the
observed inter-individual variability was the fixed, inherent, biological variability of the individuals, and not the precision of the individual estimates. In practice this was accomplished by ensuring that the observed relative variance of the individual estimates, CE 2, was less than the fixed, inherent relative variance of the group, ICV 2, and thereby made a smaller contribution to the observed relative variance of the group, C V 2, according to the following relationship. (See ref. 42 for additional details.) CV 2 = ICV 2 + CE 2
IEQ. 21
For subdivisions in which there eventually were shown to be correlations between number and age, the C V 2 used in this analysis was that of the residual variance, i.e., that not explained by age. Staining f o r Senile Plaques and Neurofibrillary Tangles Single sections, taken from the midpoint along the rostralcaudal axis of the hippocampi from the each of the 17 subjects
REGIONALLY SPECIFIC LOSS OF NEURONS
Granule Cell Layer
289
Hilus
CA3-2
CA1
30•
~o 20-
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•
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•
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25
50
75
100
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75
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:;5
10
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25
50
75
1OO
0
0
~;5
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7'5
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FIG. 2. The total number of neurons (millions) in each of the five subdivisions of the hippocampal formation plotted as a function of age (years). The regression line for number versus age is shown in the two subdivisions in which there were statistically significant correlations between neuron number and age.
older than 50 years of age, were stained for senile plaques (SP) and neurofibrillary tangles (NFT) with thioflavin-S (4). A modification of the semiquantitative scale used by Kromer-Vogt et al. (21) was used to assess the presence of SPs and NFTs in six subdivisions of the hippocampal formation and in temporal neocortex. RESULTS
Neuron Numbers The total number of neurons in each subdivision of each of the 32 individuals is shown graphically in Fig. 2 as a function of age. There were statistically significant negative correlations between neuron number and age in the hilus and subiculum (Table 1), representing 31% and 52% reductions, respectively, in the number of neurons in these two zones over the ages studied. In terms of the reduction in the number of neurons per decade, this translates to 89,000 or 4.2% of the neurons in the hilus and 550,000 or 7.3% of the neurons in the subiculum. There were no statistically significant correlations between age and the numbers of neurons in the dentate granule cell layer and the pyramidal cell layers of C A 3 - 2 and CA1. The most noteworthy aspect of the age-related loss of neurons is that it is confined to two of the five subdivisions of the hippocampal formation, hilus and subiculum. The estimates
of the total number of neurons in each of the subdivisions of each of the individuals included in this study are shown in Table 2.
Evaluation of Plaques and Tangles In 13 of the 17 subjects that were over the age of 50 years, there was evidence of either SP or neurofibrillary tangles NFT in the hippocampal and parahippocampal regions. The numbers of SPs and NFTs per m m 2 of the most affected parts of the six hippocampal subdivisions and, when available, temporal neocortex are shown in Table 3. On the basis of the CERAD criteria for the neuropathological diagnosis of Alzheimer's disease (AD) (28), one of the subjects was classified as a " p o s s i b l e " case of AD (Tables 2 and 3; Subject 29). The removal of this individual from the study did not affect the conclusions drawn with regard to the presence of statistically significant age-related losses of neurons in the hilus (r = - 0 . 3 9 5 , 2 p = 0.028) and the subiculum (r = - 0 . 4 3 9 , 2 p = 0.013). DISCUSSION
Age-Related Neuron Loss The data presented here provide the first evidence for an agerelated reduction in the total number of neurons in a part of the
TABLE 1 STATISTICAL PARAMETERS CHARACTERIZINGRELATIONSHIP BETWEEN TOTAL NEURON NUMBER AND AGE
Gran Hilus CA3-2 CA1 Subic
r
2p
- 0.11 -0.38 -0.21 -0.11 -0.46
0.55 0.032 0.24 0.53 0.008
b
-0.009
-0.035
N
CV
19.4 1.78 2.82 14.4 6.16
0.28 0.31 0.23 0.26 0.27
UCV
% Lost
0.29
31%
0.24
52%
At the end of the analysis it was confirmed, for each subdivision, that the major factor contributing to the observed relative variance of the group, CV 2, was the fixed, inherent variance of the group, ICV 2, and not the variance of the individual estimates, CE 2, which is related to the precision of the stereological procedures and the amount of sampling (42). In subdivisions in which there were eventually shown to be correlations between number and age, the residual variance, UCV 2, i.e., that not explained by age, was used instead of the CV 2. In the five subdivisions, the fixed, inherent variance of the group, 1CV 2, comprised 85%, 70%, 68%, 84%, and 85%, respectively, of the observed relative variance of the group (see Eq. 2). r = Pearson product moment correlation coefficient; 2p = significance level; b = regression coefficient, millions of neurons per year; N = mean total number of neurons in millions (n = 32); CV = coefficient of variation, (SD)/(mean); UCV = relative variation of total number, N (n = 32), (SD)/(mean), unexplained by age; % lost = based on regression value at age 13 years.
290
WEST
TABLE 2 TOTAL NUMBER OF NEURONS (MILLIONS) IN EACH OF THE SUBDIVISIONS OF THE HIPPOCAMPAL FORMATION OF 32 SUBJECTS OF VARYING AGE Subject
Age
Gran
Hilus
CA3 2
CAI
Sub
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
13 15 19 21 21 21 28 34 38 41 42 43 43 44 50 52 56 56 58 58 63 64 66 68 73 78 78 78 80 80 84 85
19.95 16.69 19.38 23.98 11.21 15.02 29.08 14.29 25.48 27.88 19.55 16.18 31.40 20.17 18.50 12.75 15.51 10.88 18.12 21.03 30.57 24.80 17.61 21.27 18.44 20.47 18.44 15.75 22.26 12.20 11.77 19.26
3.24 1.82 2.18 1.86 1.70 2.78 1.66 1.59 1.33 2.35 1.63 1.42 1.60 1.98 2.39 1.16 1.68 1.71 1.93 2.02 2.58 1.54 1.17 0.81 1.36 0.74 1.29 1.47 1.83 1.84 1.67 2.51
4.07 2.52 3.55 2.00 3.07 2.36 3.32 2.62 2.66 2.08 2.23 4.21 2.67 3.33 2.83 2.95 3.64 3.00 2.25 3.32 3.75 3.14 2.25 2.13 2.64 1.45 3.37 2.44 2.53 2.00 2.46 3.44
19.05 11.83 15.50 13.67 9.91 22.88 15.57 10.53 12.64 11.22 19.80 13.79 16.85 11.77 12.84 7.37 9.38 15.56 21.04 20.96 17.17 16.13 12.59 10.18 17.61 12.38 I 1.77 15.13 14.58 I 1.84 16.11 11.42
5.97 5.61 7.44 6.81 7.93 6.80 10.19 7.52 7.83 3.54 6.48 5.97 6.71 8.36 7.64 4.59 5.73 5.46 6.84 4.67 5.56 8.08 6.36 3.87 7.99 2.89 5.23 6.84 4.54 4.68 5.83 3.08
formation o f y o u n g e r generations, rather than n e u r o n loss. T h e r e is, at present, no w a y to formally d i s c o u n t this interpretation, in that it w o u l d require the analysis o f material collected over the past or next 72 years. O n e reason for rejecting this interpretation, however, is that the correlation b e t w e e n age and n e u r o n n u m b e r was observed in only two o f the subdivisions. Secular c h a n g e s , like intraspecific differences, are m o s t c o m m o n l y associated with differences in the overall size o f individuals and w o u l d be expected to be a c c o m p a n i e d by geometrical scaling o f brain c o m p o n e n t s , i.e., the parts o f the brain would scale in constant proportion to each other (15).
The Presence of AD T h e presence o f plaques and tangles in the h i p p o c a m p u s and temporal cortex o f a majority o f the older subjects s u g g e s t s that A D m a y be responsible the age-related losses reported here. There are, h o w e v e r , several r e a s o n s for not adopting a strict stance with regard to the elimination o f individuals with e v i d e n c e o f plaques TABLE 3 EVALUATION OF NEUROFIBRILLARY TANGLES (NFT) AND SENILE PLAQUES (SP) Subject 16 17 18 19 20 21 22 23
h u m a n brain k n o w n to be involved in m e m o r y processes that decline with age. T h e loss o f n e u r o n s in the hilus and s u b i c u l u m thereby qualify as potential m o r p h o l o g i c a l correlates o f s e n e s c e n t decline in relational m e m o r y . A l t h o u g h o n g o i n g studies d e s i g n e d to analyze the covariation b e t w e e n n e u r o n n u m b e r and perform a n c e on relational m e m o r y tasks m u s t be c o m p l e t e d before drawing definitive c o n c l u s i o n s about the relationship b e t w e e n these two p h e n o m e n a , several points c a n be m a d e to e m p h a s i z e the functional implications o f these losses and the perspectives that the regional selectivity o f the losses h a v e for the identification o f the m e c h a n i s m s involved. Prior to doing so, h o w e v e r , it is appropriate to discuss w h y secular c h a n g e s and the presence o f A D are improbable explanations o f the age-related reductions in neuron n u m ber o b s e r v e d here.
24 25 26 27 28 29 30 31
Secular Changes B e c a u s e the material u s e d in this s t u d y is a cross-sectional s a m p l e o f the entire a g e r a n g e that w a s collected over a 3-year period, it is possible that secular c h a n g e s rather than selective, regional, age-related, n e u r o n losses are responsible for the agerelated differences in the n u m b e r s o f n e u r o n s in the hilus and s u b i c u l u m . T h a t is, the differences could reflect a consistent trend toward m o r e n e u r o n s in specific s u b d i v i s i o n s o f the h i p p o c a m p a l
32
PHG NIT SP NFT SP NFT SP NFT SP NFT SP NFT SP NFT SP NIT SP NFf SP NFI" SP NFT SP NFf SP NFT SP NFT SP NFT SP NF'r SP NFT SP
0 0 0 0 + ++ + 0 +* 0 0 0 0* 0 + 0 + 0 +* +-+ + + + +* ++ + +* 0 + + +* ++ 0 0 +* 0 + + +
DG Hil CA3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CAI
Sub
0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + + 0 + + + + + 0 0 0 + +
0 0 0 0 0 ++ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + + 0 + + + ++ + + + + + 0 +
Ctx 0 0 0 0 0 0 0 -+ + 0 +++ + + +++ 0 0 -
PHG = parahippocampal gyms; DG = dentate gyms; Hil = dentate hilus; CA3 = CA3; CA1 = CA1; Sub = subiculum; Ctx = temporal neucortex; 0 = 0 NFT or SP/mm2; + = 1-5 NFT or SP/mm2; + + = 6--10NFTorSP/mm2; + + + = 11-25NFrorSP/mm2; ++++ = >25 NF'I" or SP/mm2; * = entorhinal cortex; - = no observations.
REGIONALLY SPECIFIC LOSS OF NEURONS and tangles from this study of normal aging. First and foremost, the relationship between normal aging processes and the processes involved in the formation of plaques and tangles is not fully understood. Both plaques and tangles are observed in nondemented individuals (4,40) and are not unequivocal evidence of AD. The neuropathological criteria recommended by NINCDS-ADRDA and CERAD, for a neuropathological diagnosis of AD, both involve age-adjusted plaque counts (20,28), indicating that, by definition, the mere presence of plaques is not adequate for the diagnosis. Although the criteria themselves are the subject of debate (13), primarily as a consequence of the difficulties encountered when establishing threshold values for a neuropathological diagnosis and the poor correlation between the severity dementia and number of SPs (3), it could be argued that the one possible case (Tables 2 and 3; Subject 29) should be excluded from this study on the basis of neuropathological criteria. The removal of this case, however, does not alter the main conclusion drawn from this study, i.e., that there are statistically significant age-related losses of neurons in the hilus and the subiculum. There are two reasons for not excluding any of the subjects from this study on the basis of suspected AD. First, the clinical criteria, which are used in concert with the neuropathological criteria when diagnosing AD according to the CERAD and NINCDS-ADRDA guidelines, are not fully satisfied by any of the subjects used in this study. Although they were not explicitly tested psychometrically for signs of dementia, there was no evidence in their medical records of neurological or psychiatric disorders. All died under relatively acute circumstances involving little or no hospitalization and apparently conducted normal lives prior to their deaths. Second, the pattern of neuron loss reported here is qualitatively different from that observed in advanced cases of AD. Preliminary evaluations of the regional neuron loss in advanced cases of AD indicates that it is the CA1 region, and not the hilus or the subiculum, that selectively looses neurons in AD patients that meet both clinical and neuropathological criteria (44). The presence of SPs and NFFs in 13 of the 17 of the subjects over the age of 50 years does underline both the practical problems involved in obtaining aged subjects who are free of the pathological hallmarks of AD and the uncertainty surrounding the concept of normal, pathology free, aging. The final resolution of the issue as to when individuals with plaques and tangles should be excluded from a study of normal aging awaits a more complete understanding of the relationship between normal aging processes and the processes involved in the formation of plaques and tangles. With this caveat in mind, along with that with regard to the secular interpretation of the data described above, the data will be discussed exclusively in terms of neuron loss related to normal aging.
Functional Implications of the Neuron Loss In view of what is known about the functional organization of hippocampal neurons, the reported losses can be expected to reduce the information processing capacity of the hippocampal formarion. The hippocampal subdivisions are connected by a unidirectional system of parallel and serial neuronal circuits through which information flows from the dentate gyms to the subiculum (2) (Fig. 1C). The neurons in the hilus participate in the flow of information though the initial series of circuits in the hippocampal formation. A reduction in the number of these neurons would be expected to reduce the associative capacity of the dentate granule cells and the amount or resolution of the information sent from this region to CA1 (7,23,31). The loss in subiculum may represent a more profound deficit because of the relative magnitude of the neuron loss and its position at the end of the unidirectional system
291 of fiber connections within the hippocampal formation. There it serves as the major distributor of hippocampal output information to other cortical regions, including entorhinal cortex (45), that are intimately involved in the "back projections" required to establish long term memories (34,39). Although it has been suggested that age-related neuron loss can be viewed as a continuation of normal developmental processes that fine tunes neuronal circuitry and may even have functional benefits (24), there are limits to the degree to which a reduction in the number of neurons within specific subdivisions of the hippocampal formation can be construed as beneficial. Disruptions in the flow of information through the hippocampal formation resulting from regional neuron loss produce anteriograde amnesia in humans (46) and deficits in performance on specific relational memory tasks in experimental animals (29,47). A determination of the point at which neuron loss translates to functional impairment must await the results of ongoing studies in humans and nonhuman primates designed to evaluate the covariation between the number of neurons in specific hippocampal subdivisions and performance on relational memory tests. The results of these studies will also be helpful in determining the degrees to which compensatory physiological, structural, and biochemical processes, both inside and outside the hippocampal formation are involved in senescent decline in relational memories.
Regional Selectivity The regional selectivity of the age-related neuron loss reported here may provide clues to the mechanisms involved in the loss and maintenance of specific groups of neurons during aging. Promising directions for future research include more detailed examinations of the relationship between neuron loss and Alz-50 immunoreactivity (8), acetlycholinesterase-rich neurons that develop later in life (25), and the regional distribution of receptor systems that mediate excitatory synaptic transmission (11). The latter is of particular interest because specific subgroups of these receptors have been shown to be involved in excitotoxic neuron death that is associated with a number of pathological states and can be potentiated by stress and glucocorticosteroids (36). The age-related loss of neurons observed in the hilus warrants additional study to determine whether the specific subgroups of hilar neurons, reported to be involved in the initial pathological changes related to ischemia (19), epilepsy (12,37,38), and those associated with AD (9) contribute disproportionately to the observed loss and provide evidence of the participation of neurodegenerative mechanisms related to these other phenomena. The regional pattern of age-related neuron loss is similar but not identical to that seen in the hippocampal formation of patients with AD. Qualitative and quantitative studies clearly indicate that the CA1 region is one of the most heavily afflicted regions in AD patients (6,41) and undergoes extensive neuron loss in advanced cases (44). In spite of similarities in the losses of neurons in the hilus and the subiculum of normal-aged subjects and AD patients, the lack of evidence of an age-related change in the number of CA1 neurons, in the data presented here, strongly suggests that there is a fundamental qualitative difference between the regional pattern of neuron loss associated with normal aging and that associated with AD and a fundamental difference in the mechanisms (22) involved in the neuron loss associated with the two processes. ACKNOWLEDGEMENTS I thank Dr. Bradley Hyman (Neurology Service, Massachusetts General Hospital, Harvard Medical School, Boston, MA) for his assistance with the staining and evaluation of the thioflavine-S preparations. Also, I thank Maj-Britt Lundorf for assistance with the histology; Anette Larsen
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and Albert Meier for assistance with the illustrations; the Departments of Pathology at Aarhus Kommunehospitalet and Aarhus Amtssyghus, the Department of Forensic Medicine, Aarhus University, and The Neurological Research Laboratory, Bartholin Institute, Copenhagen for providing the histological material used in this study; Henrik Clausen for his roll in the development of the GRID stereological software package; Erik MOiler
of the Danish office of Leica, Glostrup, Denmark for assistance with the microscope equipment used for the stereological analysis. This work has been supported by The Danish Health Sciences Research Council, Aarhus University Research Foundation, Fonden til Forskning af Sindslidelser, Fonden af 1870, and the Danish Foundation for Experimental Research in Neurology.
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