Patients with vascular damage due to microvascular pathology have significant hippocampal neuronal loss

Abstract

Alzheimer's disease (AD) is characterised by functional impairment, cerebral atrophy, and degeneration of specific neuronal populations, especially pyramidal neurones of the cerebral cortex and hippocampal formation. Although patients with subcortical vascular dementia have been shown to have similar metabolic and volumetric deficits to those with AD, the underlying pathogenesis of these changes is poorly understood.
To determine whether pyramidal cell loss occurs in small vessel disease (SVD) dementia by quantifying hippocampal volume and CA1 neurone number.
Fifty four prospectively studied patients with dementia were screened, and four patients fulfilling criteria for SVD with no other significant neuropathological abnormality were identified. These were compared with five patients fulfilling criteria for AD and seven controls matched for age and sex. The hippocampal formation was serially sectioned, and the number of CA1 pyramidal neurones estimated using the optical dissector technique. Analysis of variance was used to evaluate group differences.
Patients in both the AD and SVD groups showed a substantial loss of pyramidal neurones from the CA1 region. The pattern of hippocampal atrophy and the degree of CA1 neuronal loss were similar in the two dementia groups.
These findings support recent in vivo studies showing similar metabolic deficits and atrophy in AD and subcortical vascular dementia. In addition, they provide evidence that the underlying cause of these abnormalities is a similar loss of neurones. Whereas the cause of the neuronal loss in AD is related to the deposition of abnormal proteins, the cause in SVD is unknown. In the absence of other pathologies, damage to cerebral microvasculature should be considered a likely candidate.

Full text

PAPER
Patients with vascular dementia due to microvascular
pathology have significant hippocampal neuronal loss
J J Kril, S Patel, A J Harding, G M Halliday
.............................................................................................................................
J Neurol Neurosurg Psychiatry
2002;72:747–751
Background: Alzheimer’s disease (AD) is characterised by functional impairment, cerebral atrophy,
and degeneration of specific neuronal populations, especially pyramidal neurones of the cerebral cor-
tex and hippocampal formation. Although patients with subcortical vascular dementia have been
shown to have similar metabolic and volumetric deficits to those with AD, the underlying pathogenesis
of these changes is poorly understood.
Objective: To determine whether pyramidal cell loss occurs in small vessel disease (SVD) dementia by
quantifying hippocampal volume and CA1 neurone number.
Methods: Fifty four prospectively studied patients with dementia were screened, and four patients ful-
filling criteria for SVD with no other significant neuropathological abnormality were identified. These
were compared with five patients fulfilling criteria for AD and seven controls matched for age and sex.
The hippocampal formation was serially sectioned, and the number of CA1 pyramidal neurones esti-
mated using the optical dissector technique. Analysis of variance was used to evaluate group
differences.
Results: Patients in both the AD and SVD groups showed a substantial loss of pyramidal neurones from
the CA1 region. The pattern of hippocampal atrophy and the degree of CA1 neuronal loss were simi-
lar in the two dementia groups.
Conclusions: These findings support recent in vivo studies showing similar metabolic deficits and atro-
phy in AD and subcortical vascular dementia. In addition, they provide evidence that the underlying
cause of these abnormalities is a similar loss of neurones. Whereas the cause of the neuronal loss in
AD is related to the deposition of abnormal proteins, the cause in SVD is unknown. In the absence of
other pathologies, damage to cerebral microvasculature should be considered a likely candidate.
Vascular dementia is recognised as one of the most com-
mon causes of dementia after Alzheimer’s disease (AD).
Traditionally, vascular dementia has been distinguished
from AD by the pattern of clinical progression, with vascular
dementia characterised by a stepwise rather than a slowly
progressive course of deterioration. However, this view
appears overly simplistic, as clinical presentations can vary
enormously in patients with vascular dementia.1–5 In particu-
lar, some cases of vascular dementia are clinically indistin-
guishable from AD as they show no evidence of a stepwise
deterioration.2At autopsy, these patients have small vessel
disease (SVD), leucoencephalopathy, and microscopic infarc-
tion rather than the pathology of AD.2This has been confirmed
in a carefully selected autopsy series, leading Esiri and
colleagues4to suggest that microvascular disease, rather than
macroscopic infarction, is the most common pathology
underlying vascular dementia.
Although SVD is being increasingly recognised as a subtype
of vascular dementia,56no validated criteria for the identifica-
tion of such cases, either clinically or pathologically, have been
established. At present, the pathological classification of SVD
is largely descriptive and often relies on the pathologist’s
opinion of whether the vascular pathology contributed to the
dementia.7The study by Esiri and colleagues4proposed a
semiquantitative grading scale for SVD using the presence and
increasing severity of a number of features of microvascular
pathology.The systematic use of this scale may lead to a better
understanding of the mechanisms by which SVD causes, or
contributes to, dementia.
An increase in SVD is known to occur with age and in the
presence of cerebrovascular risk factors.8In non-demented
persons, microvascular pathology influences specific cognitive
functions such as those involving speed of mental
processing.8Although the substrate for dementia in SVD is
still unclear, in vivo studies910indicate a similar pattern and
degree of cortical dysfunction and volume loss in AD and SVD.
The functional deficits reported to date in patients with SVD
dementia suggest significant cortical and limbic degeneration,
similar to AD, although the underlying pathology is predomi-
nantly white matter leucoencephalopathy. Remote hippocam-
pal injury has been found in some cases with extensive micro-
vascular disease,11 but has not been systematically studied.
Indeed, there have been no studies on the distribution and
extent of neuronal loss in patients with SVD. In this study,
neuronal loss from the CA1 sector of the hippocampus was
quantified in demented patients who did not reach pathologi-
cal criteria for AD, or any other neurodegenerative disease,but
who had significant SVD. The aim of this study was to deter-
mine if SVD per se can cause hippocampal degeneration and
as such contribute to the dementia syndrome.
MATERIALS AND METHODS
Case selection and characterisation
Cases were collected from both population based research
studies on AD and from a tertiary referral centre for neuro-
degenerative diseases. This study was approved by the ethics
review committees of the Central and South Eastern Sydney
Area Health Services. All patients were examined by a
neurologist or geriatrician. Additional corroborative infor-
mation for each patient was obtained from an informant
interview to ascertain the pattern and type of deficits and to
.............................................................
Abbreviations: AD, Alzheimer’s disease; SVD, small vessel disease
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr J J Kril, Centre for
Education and Research on
Ageing, Concord Hospital,
Concord NSW 2139,
Australia;
jilliank@med.usyd.edu.au
Received
12 November 2001
Accepted
15 January 2002
.......................
747
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aid in the estimation of disease duration. Fifty four patients
who met NINCDS-ADRDA criteria for possible or probable
AD12 came to autopsy during the collection period of seven
years.
Brains were fixed in neutral buffered formalin for two
weeks, and then the weight, volume, and anteroposterior
length of the cerebral hemispheres determined.13 The hemi-
spheres were then sectioned at about 3 mm intervals in the
coronal plane. The cerebral cortex, hippocampus, brainstem,
and cerebellum were sampled from standardised regions of
each brain. Sections were stained with haematoxylin and
eosin, modified Bielschowsky silver and ubiquitin immuno-
histochemistry, and examined to perform the classifications
outlined below and to exclude pathologies other than AD and
SVD.
Each case was given a grading using each of the following
classification schema.
(a) CERAD14: normal, possible, probable, or definite AD.
Neuritic plaques were evaluated in sections of the temporal
neocortex (Brodmann area 20) stained with the modified
Bielschowsky silver stain. Ten random microscope fields at
100×magnification graded as having none (<1/field), rare
(2–5/field), mild (6–15/field), moderate (16–50/field), or
severe (>50/field) numbers of neuritic plaques.
(b) Braak15 16: normal (stages 0–2), transitional limbic (stages
3 or 4), or neocortical (stages 5 or 6) AD. Neurofibrillary tan-
gles were evaluated in sections of the hippocampal formation
and temporal neocortex stained with the modified Biels-
chowsky silver stain. Ten random microscope fields at 200×
magnification graded into none (nil found), rare (<1/field),
mild (1–2/field), moderate (3–10/field) or severe (>10/field)
numbers of neurofibrillary tangles.
(c) Esiri4: normal (grades 0 or 1), SVD (grades 2 or 3). Sections
from the dorsolateral prefrontal association cortex (Brod-
mann area 9), anterior cingulate gyrus (Brodmann area 24),
and primary motor cortex (Brodmann area 4) were stained
with haematoxylin and eosin. Briefly, cases were classified as
normal if they exhibited little or no widening of perivascular
spaces, thickening of vascular walls, and/or a few perivascular
macrophages. Notably, myelin pallor, attenuation of nerve
fibres, and gliosis were absent. Cases were classified as SVD
when widening of perivascular spaces (fig 1C), thickening of
vascular walls (fig 1A), accumulation of perivascular macro-
phages (fig 1B), and perivascular myelin pallor or attenuation
of nerve fibres (fig 1D) were present.
For this study, case classification for SVD was based on the
presence of SVD using the Esiri grading scheme and the
absence of probable or definite CERAD AD or a Braak neocor-
tical neuritic stage of AD. Four of the 54 cases fulfilled such
criteria for SVD in the absence of any other significant degen-
erative condition. For comparison, five AD cases were selected
on the basis of absence of SVD and the presence of definite
CERAD AD and a Braak neocortical stage of AD (table 1).
Although 12 of the 54 cases showed both SVD and AD using
the classifications outlined above (four with additional large
vessel infarction, two with dementia with Lewy bodies,17 and
two with hippocampal sclerosis), these cases were not
included in the study. This enabled examination of the role of
SVD, and its comparison with AD, without the complication of
an overlap in the type of cortical pathology in the two demen-
tia groups. Seven controls matched for age and sex from the
population based research studies who were without signifi-
cant neuropathology (normal using all schema) were also
selected for comparison.
Quantification of hippocampal formation
The techniques used to quantify hippocampal volume and
CA1 neurone number have been described in detail in our
previous publications.18 19 Briefly, the entire hippocampal
formation was dissected from the right hemisphere, the blocks
cryoprotected in 30% sucrose, and three 48 µm thick sections
cut from the caudal face of each block on a freezing
microtome. Series of sections from each case were stained
Figure 1 Photomicrographs showing white matter pathology used in the grading of small vessel disease pathology. (A) Haematoxylin and
eosin stained section through the frontal white matter showing an arteriole with a thickened vessel wall. Scale equivalent to (B). (B)
Haematoxylin and eosin stained section through the frontal white matter showing haemosiderin laden macrophages around a white matter
blood vessel. (C) Haematoxylin and eosin stained section through the frontal white matter showing perivascular dilatation with attenuation of
surrounding white matter. (D) Modified Bielschowsky silver stained section through the frontal white matter showing attenuation of myelin in the
deep white matter.
748 Kril, Patel, Harding, et al
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with haematoxylin and eosin, cresyl violet, and nickel
peroxidase.20 The boundaries of each of the hippocampal sub-
regions were delineated on the cresyl violet stained sections
and drawn after magnification on a microfiche reader (×19
magnification). The volume of each region was then
determined using point counting,18 19 and the total number of
neurones in the CA1 region estimated using the optical
dissector technique.18 19 21 22 The first dissector frame (120 µm ×
120 µm) was placed randomly in the CA1 sector,and then sys-
tematic samples at 2.4 mm horizontally and 1.2 mm vertically
were counted. The coefficient of variance for the control group
was 0.208, and the coefficient of error was 0.074.
Group means were compared using analysis of variance,
and correlations examined using linear regression analysis
(Statview 5, SAS Institute Inc, Cary, North Carolina, USA).
RESULTS
Case descriptions
Table 2 gives the details of the classification for each case. The
mean age did not differ between any of the groups (F=2.61;
p=0.112), although patients in the SVD group were on
average older than those in the AD groups (86 (12) years v76
(4) years; controls had an mean age of 75 (8) years) (values
are mean (SD)).
Summaries of the clinical presentation and progression of
each case with SVD are listed below to illustrate more clearly
the type of case studied.
S1 was a 69 year old woman who presented with increasing
impairment of memory. Her past medical history consisted of
type II diabetes, hyperlipidaemia, and hypertension, which
was controlled by captopril. After evaluation she was
diagnosed with AD. The following year she suffered a stroke-
like episode, but computed tomography showed no focal
lesion. Cerebral atrophy and leucoaraiosis were described. She
continued to decline and was cared for at home by her
husband. Three years after presentation she was admitted to a
regional hospital after a fall. She died nine days later after a
five day history of decline in neurological function. Death was
attributed to an acute myocardial infarct. Her neuropathologi-
cal examination showeda3mmlacune in the right putamen
and an area of destruction of the white matter at the angle of
the right lateral ventricle.
S2 was a university professor who retired at 65 years of age,
although had continued to participate in academic activities.
He presented at age 75 with a two year history of increasing
difficulty with word finding and had ceased lecturing because
of this. He had also developed difficulties with navigation
while driving. Formal assessment at this time revealed a mini
mental state examination23 of 30/35, and a diagnosis of AD was
made. He had no history of diabetes or hypertension, although
he had suffered a transient ischaemic attack two years earlier.
His medications were aspirin, melleril, and voltaren. Over the
ensuing three years he deteriorated, became inert, and was
unable to handle money, although he was able to dress and
groom himself. Neurological examination showed brady-
kinesia, rigidity, and ataxia. He died at age 80, and
neuropathological examination showed small lacunes in the
left internal capsule, right claustrum, and right globus
pallidus. There was some loss of pigmented neurones from the
substantia nigra but no Lewy bodies.
S3 was a 93 year old woman referred for evaluation. She had
a 10 year history of a decline in domestic duties and increas-
ing neglect of her self care. She had delusions and wandered
if left unattended. On examination, she had deficits in
memory, insight, visuospatial abilities, drive, and planning.
Her mini mental state examination was 14/30. Her previous
history included a myocardial infarct eight years earlier, but
was otherwise unremarkable. She had several admissions to
hospital after falls, including a fractured pelvis. She was
admitted to a nursing home one year before death.
Neuropathological examination showed an old cavitated
infarct involving the internal capsule, caudate, and putamen,
which measured 1.5 cm in anterioposterior extent. Haemo-
siderin laden macrophages were present at this site.
S4 was a 94 year old woman who presented with a one year
history of increasing forgetfulness. She had a sustained delu-
sion that her symptoms were due to her use of a pesticide
spray. She also had visual hallucinations. Investigation
Table 1 Screening procedure and classification
schema
Diagnosis CERAD14 Braak16 Esiri4
SVD alone Normal or possible Normal (0–2) SVD (2–3)
or limbic (3–4)
AD alone Probable or definite Neocortical (5–6) Normal (0–1)
All cases met NINCDS-ADRDA34 clinical criteria for possible or
probable AD. Note the lack of overlap between the two groups. The
combination of the CERAD and Braak schemes for AD is consistent
with current recommendations for the diagnosis of AD.35
SVD, Small vessel disease; AD, Alzheimer’s disease.
Table 2 Age, sex, and neuropathological grading for each case
Case
Age
(years) Sex
Duration
(years)
Plaque
density CERAD*
NFT density
in ctx Braak*
SVD*
grade
S1 73 F 4 6–15 Possible 0 Normal 3
S2 80 M 7 Rare Normal Rare Limbic 3
S3 96 F 13 Rare Normal 1–2 Limbic 3
S4 97 F 4 6–15 Possible Rare Limbic 2
A5 69 F 4 16–50 Definite >10 Neocortical 1
A6 70 F 7 >50 Definite >10 Neocortical 1
A7 76 M 4 >50 Definite >10 Neocortical 0
A8 78 M 14 16–50 Definite >10 Neocortical 1
A9 81 F 5 16–50 Definite 3–10 Neocortical 0
C1 61 F – Rare Normal 0 Normal 1
C2 69 F – Rare Normal 0 Normal 0
C3 72 M – Rare Normal 0 Normal 0
C4 74 M – Rare Normal 0 Normal 0
C5 79 M – Rare Normal 0 Normal 1
C6 84 F – Rare Normal 0 Normal 1
C7 85 M – Rare Normal 0 Normal 1
*CERAD14; Braak16; Esiri.4
Ctx, Cortex; NFT, neurofibrillary tangles; S, SVD dementia; A, Alzheimer’s disease; C, non-demented control.
Hippocampal neuronal loss in vascular dementia 749
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showed deficits in memory, praxis, and an unsteady gait con-
sistent with having had bilateral knee replacements. She had
no history of diabetes, cardiac disease, or hypertension and
was taking no medicines. She died from a cardiac arrest while
resident in a nursing home. Neuropathological examination
showed generalised atrophy, with no focal lesions.
Diagnostic pathology
Figure 1 shows examples of the white matter pathology in the
SVD cases. Changes included both those involving the vessels
themselves and those involving the surrounding tissue. Thick-
ening of arteriolar walls (fig 1A), accumulation of macro-
phages (fig 1B), perivascular dilatation (etat criblé, fig 1C),
and attenuation of myelin (fig 1D) were seen in all cases.
These changes were observed in all of the white matter regions
examined.
In the SVD cases, no cortical plaques were detected in two
cases and moderate numbers were found in the other two
(table 2). Cortical neurofibrillary tangles were absent in three
cases and mild in the fourth (table 2). In contrast, the patients
with AD had severe cortical neurofibrillary tangles in four of
the five cases and moderate in the fifth (table 2). Cortical
plaques were severe in two of the five and moderate in the
remainder (table 2).
Hippocampal volume changes
The grey matter component of the hippocampal formation is
significantly smaller in both of the dementia groups than in
the control group (table 3). When the subregion volumes were
analysed, all regions except the dentate gyrus and hilus (CA4)
were smaller in patients with AD than in controls. In SVD, sig-
nificant atrophy was confined to the large CA1 and subiculum
grey matter regions (table 3). The magnitude of the atrophy in
these hippocampal regions was not significantly different
between the dementia groups.
CA1 neurone number
The total estimated neurone number in the CA1 is also
significantly reduced in both dementia groups compared with
controls (F=17.3; p=0.0002; fig 2).The magnitude of CA1 loss
is not significantly different between the SVD and AD groups
(50% and 69% respectively). Considerable variation in
neurone number exists within the two dementia groups,
although this is not as a result of disease duration (r2=0.035;
p=0.63) or age at death (r2=0.001; p=0.94)
DISCUSSION
Significant atrophy and neuronal loss from the CA1 region of
the hippocampus are one of the pathological hallmarks of
AD.22 24 Interestingly in this study we have shown that a simi-
lar pattern and magnitude of hippocampal atrophy and
neurone loss also occurs in patients who do not reach patho-
logical criteria for AD but who have SVD. These findings,
together with the fact that all nine patients with dementia
met clinical criteria for probable AD, suggest that SVD can
result in a cortical dementia syndrome similar to AD and that
hippocampal neuronal loss contributes to this clinical picture.
Hippocampal damage in AD is considered an early event
with substantial pathology and early atrophy preceding symp-
tom onset.25 26 After these early changes, the widespread depo-
sition of abnormal proteins within the association cortices
occurs, with cognitive decline leading to clinical dementia and
appreciable neurodegeneration.16 27 28 This knowledge of the
progression of pathology underlying the clinical dementia and
cortical degeneration of AD has been derived from studying a
large number of cases. Similar studies have not been
performed for SVD dementia because of the lack of consensus
on diagnostic requirements for SVD and the relative rareness
of cases in which SVD is the only substrate for the dementia.
In our prospectively studied series of patients with dementia,
and other autopsy series,24 the proportion of cases in which
significant microvascular disease is the principal pathology is
small (<10% in this study). Several studies have investigated
the consequences of coexisting cerebrovascular disease and
AD. Both the OPTIMA and Nun studies show that cognitive
performance is worse in subjects with early AD and cerebro-
vascular disease (both large vessel and small vessel disease)
than in patients with AD alone,29 30 suggesting that the vascu-
lar pathology contributes to dementia severity. Similarly, in
subjects with equivalent cognitive performance, the burden of
AD-type pathology is less in those with AD and coexisting
cerebrovascular disease than in those with AD alone,731 also
supporting the concept that vascular pathology contributes to
the dementia. Our results suggest that this contribution is due
to pyramidal cell loss through non-AD mechanisms.
The study of vascular dementia has been dominated by
large vessel disease (infarction) and only recently has the
importance of SVD been realised.411The loss of neurones with
large vessel disease is unquestioned, although neuronal loss
due to microvascular pathology has been anecdotal and not
systematically studied. Our study not only provides evidence
for the existence of SVD as an independent cause of dementia,
but also provides some insights into the pathogenesis of this
dementia by showing that substantial hippocampal pyramidal
cell loss occurs in SVD. This suggests a causative link between
Table 3 Volumes (mm3) of the hippocampal subregions for each group
SVD p Value AD p Value Control
Total 1302 (142) 0.0053 1035 (74) 0.0001 1835 (108)
CA1 466 (77) 0.0270 317 (46) 0.0004 662 (46)
CA2–3 119 (10) 0.5813 89 (14) 0.0344 129 (12)
CA4 160 (8) 0.4187 158 (12) 0.3516 185 (25)
Dentate gyrus 51 (2) 0.6050 59 (5) 0.0924 47 (4)
Subiculum 254 (37) 0.0112 211 (23) 0.0025 490 (66)
Presubiculum 251 (36) 0.0668 201 (26) 0.0028 322 (17)
Values are mean (SEM). Values significantly different from controls are shown in bold.
SVD, Small vessel disease; AD, Alzheimer’s disease.
Figure 2 Neurone number in the CA1 region of the hippocampus
in control, Alzheimer’s disease (AD) and small vessel disease (SVD)
groups. Bar = mean ±1 SD, p values for AD and SVD groups
relative to control group.
8
7
6
5
4
3
2
1
0Control AD SVD
p = 0.0029
p < 0.0001
10–6 × Total CA1 neurone number
750 Kril, Patel, Harding, et al
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SVD, pyramidal cell loss, and hippocampal degeneration, and
raises the possibility that cortical microvascular pathology
contributes to dementia through pyramidal cell loss. In vivo
studies showing a similar pattern and severity of cortical dys-
function and atrophy in AD and SVD support this
suggestion.910 Overall, there may be little difference in the
degree of neuronal loss between SVD and AD, with only the
mechanism of degeneration differing between these dementia
syndromes.
The mechanism of neuronal degeneration in SVD may be
apoptosis, consistent with an absence of pathological debris.
Apoptosis is increased in ischaemia,32 and markers of apopto-
sis are increased in areas of leucoaraiosis compared with adja-
cent white matter.33 An increase in cell cycle proteins within
hippocampal neurones in patients with SVD31 is also
consistent with abnormal cellular processing leading to apop-
tosis. The location of these changes within hippocampal CA1
neurones suggests they may precede the cell loss shown in this
study. It is now important to study large cohorts of patients
with SVD in order to determine the temporal sequence of the
neuronal damage shown and the onset of dementia. Such
knowledge is essential for effective treatment strategies.
ACKNOWLEDGEMENTS
This work was supported by a programme grant from the Medical
Foundation of the University of Sydney and by the National Health
and Medical Research Council of Australia. JJK was a Medical Foun-
dation Fellow, and GMH is a NHMRC Principal Research Fellow.
.....................
Authors’ affiliations
JJKril,SPatel,Centre for Education and Research on Ageing,
Department of Medicine, University of Sydney, Sydney, NSW, Australia
J J Kril, Department of Pathology, University of Sydney
A J Harding, G M Halliday, Prince of Wales Medical Research Institute
and the University of New South Wales, Australia
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