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Neurobiology of Aging 36 (2015) 1075e1082

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Neurobiology of Aging
journal homepage: www.elsevier.com/locate/neuaging

Subcortical structures in amyotrophic lateral sclerosis


Henk-Jan Westeneng a, Esther Verstraete a, Rene Walhout a, Ruben Schmidt a,
Jeroen Hendrikse b, Jan H. Veldink a, Martijn P. van den Heuvel c,1,
Leonard H. van den Berg a, *,1
a

Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands
Department of Radiology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands
c
Department of Psychiatry, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 12 June 2014
Received in revised form 14 August 2014
Accepted 1 September 2014
Available online 6 September 2014

The aim of this study was to assess the involvement of deep gray matter, hippocampal subelds, and
ventricular changes in patients with amyotrophic lateral sclerosis (ALS). A total of 112 ALS patients and
60 healthy subjects participated. High-resolution T1-weighted images were acquired using a 3T MRI
scanner. Thirty-nine patients underwent a follow-up scan. Volumetric and shape analyses of subcortical
structures were performed, measures were correlated with clinical parameters, and longitudinal changes
were assessed. At baseline, reduced hippocampal volumes (left: p 0.007; right: p 0.011) and larger
inferior lateral ventricles (left: p 0.013; right: p 0.041) were found in patients compared to healthy
controls. Longitudinal analyses demonstrated a signicant decrease in volume of the right cornu
ammonis 2/3 and 4/dentate gyrus and left presubiculum (p 0.002, p 0.045, p < 0.001), and a signicant increase in the ventricular volume in the lateral (left: p < 0.001; right: p < 0.001), 3rd (p < 0.001)
and 4th (p 0.001) ventricles. Larger ventricles were associated with a lower ALSFRS-R score (p 0.021).
In conclusion, ALS patients show signs of neurodegeneration of subcortical structures and ventricular
enlargement. Subcortical involvement is progressive and correlates with clinical parameters, highlighting
its role in the neurodegenerative process in ALS.
2015 Elsevier Inc. All rights reserved.

Keywords:
Amyotrophic lateral sclerosis
Magnetic resonance imaging
Longitudinal
Basal ganglia
Hippocampal subelds

1. Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive upper and lower motor neuron
degeneration (Kiernan et al., 2011). Although progressive motor
neuron degeneration is the hallmark feature of ALS, widespread
extramotor brain involvement can be found as well (Agosta et al.,
2007; Verstraete et al., 2014). It has been shown that ALS affects a
subnetwork in the brain including white matter connections with
subcortical structures such as the thalamus, caudate nucleus, putamen, globus pallidus, and hippocampus (Verstraete et al., 2014).
These ndings may suggest involvement of these structures in the
underlying neurodegenerative process in ALS.
Magnetic resonance imaging (MRI) is a noninvasive, sensitive
method allowing in vivo study of volume changes of structures
within the brain. Most neuroimaging studies have focused on the

* Corresponding author at: Department of Neurology, G03.228, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Tel.: 31 88
7557939; fax: 31 30 2542100.
E-mail address: L.H.vandenBerg@umcutrecht.nl (L.H. van den Berg).
1
Authors contributed equally.
0197-4580/$ e see front matter 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.neurobiolaging.2014.09.002

cortical surface and white matter changes, aiming to capture


upper motor neuron degeneration (Foerster et al., 2013; Turner
et al., 2012). Postmortem studies have, however, shown prominent involvement of subcortical structures in ALS (Brettschneider
et al., 2013; Geser et al., 2008; Takeda et al., 2009). In addition,
studies using positron emission tomographyecomputed tomography (PET-CT), single-photon emission computed tomography
(SPECT), magnetic resonance spectroscopy, and diffusion tensor
imaging have also shown involvement of deep gray matter (DGM;
thalamus, caudatus, putamen, pallidum, hippocampus, amygdala,
accumbens), but morphometric changes have been reported
sporadically (Sach et al., 2004; Turner et al., 2004; Verstraete
et al., 2014). Currently, there is only 1 cross-sectional study that
has reported a more detailed morphometric analysis of the DGM
in ALS (Bede et al., 2013). This cross-sectional study in 39 ALS
patients showed reduced volumes of the left caudate, left hippocampus, and right accumbens in ALS, underscoring the relevance of the subcortical structures in the disease process.
Although, in the classical conception, the hippocampus is a
structure of the cortex (3-layered cortex, not neocortex), one may
also refer to it as a subcortical structure because it is located under
the cortex.

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H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082

Longitudinal analyses are important for the understanding of


patterns of disease progression and might result in the discovery of
a more specic marker that can be used to monitor disease progression in ALS. Longitudinal neuroimaging studies may be an
important tool to assess cerebral neurodegeneration in detail in a
noninvasive way. For example, hippocampal atrophy has been reported in ALS but also in other neurological and psychiatric diseases
(Bede et al., 2013; Thompson et al., 2004). Longitudinal analyses, in
combination with a more detailed analysis of the hippocampus
(hippocampal subeld segmentations), might shed more light on
when the hippocampus becomes involved and which subelds in
particular may be affected.
We therefore studied volumes of ventricles and DGM (including
hippocampal subelds) cross-sectionally and longitudinally in a
large group of patients with ALS, and correlated our ndings with
clinical characteristics.
2. Methods
2.1. Participants
All 172 participating subjects were recruited from the outpatient
clinic for motor neuron diseases of the University Medical Center
Utrecht in The Netherlands. Patients were classied as having
denite, probable or possible ALS using the revised El Escorial
criteria after excluding other conditions (Brooks et al., 2000). Subjects with a history of brain injury, epilepsy, psychiatric illness,
other neurodegenerative disease (including frontotemporal lobe
dementia), or structural brain disease were excluded. The median
time between the rst and second MRI scan was 5.5 months.
Written informed consent was obtained from all participants, in
accordance with the Declaration of Helsinki.
2.2. Clinical parameters
Clinical characteristics, including handedness, disease duration,
and survival were recorded. Functional status was evaluated using
the revised ALS Functional Rating Scale (ALSFRS-R) (Cedarbaum
et al., 1999). The disease progression rate was calculated using the
formula (48  ALSFRS-R score)/disease duration (in months). Disease duration was evaluated from symptom onset.
2.3. Data acquisition
A 3T Philips Achieve Medical Scanner was used to acquire a
high-resolution T1-weighted image. Acquisition parameters were
as follows: 3-dimensional fast eld echo (FFE) using parallel imaging; TR/TE 10/4.6 ms, ip angel 8 , slice orientation sagittal,
0.75  0.75  0.8 mm (0.45 mm3) voxel size, eld of view (FOV)
160  240  240 mm (9.216 dm3) and reconstruction matrix
200  320  320 covering the whole brain. Acquisition time was
11 minutes. The high-resolution MRI scans for scientic research
were combined with a standard MRI examination, and these scans
were reviewed by an experienced neuroradiologist. In the case of
structural abnormalities, made scans were excluded.
2.4. Data processing
Two groups of analyses were performed: cross-sectional and
longitudinal. Both analyses focused on volumes of ventricles, DGM,
and hippocampal subelds.
For the cross-sectional analysis, subcortical volumes were
automatically segmented and measured by FreeSurfer version 5.1
(Fischl et al., 2002, 2004). In addition, hippocampal subelds were
automatically segmented and measured using a FreeSurfer

subroutine (Van Leemput et al., 2009). In this article, we have


focused on cornu ammonis 1 (CA1), CA2/3, CA4/dentate gyrus (CA4/
DG), subiculum, and presubiculum. Fimbria and hippocampal
ssure were disregarded because these are the smallest subelds
and segmentation is less reliable (Van Leemput et al., 2009).
To detect subtle regional volume changes of DGM, we performed
a shape analysis of DGM (thalamus, putamen, caudate nucleus,
nucleus accumbens, and hippocampus), previously reported to be
altered in ALS (Agosta et al., 2009; Bede et al., 2013; Chang et al.,
2005; Thivard et al., 2007). The shape of the above-mentioned
DGM (except the hippocampus, the subelds of which were studied) was analyzed using the FSLs FIRST module version 5.0.0
(Patenaude et al., 2011). This vertex-based shape analysis was corrected for multiple testing using permutation tests with 10,000
permutations of cluster mass.
The longitudinal analysis was performed using FreeSurfer, which
creates an unbiased within-subject template space and image using
robust inverse consistent registration (Reuter and Fischl, 2011;
Reuter et al., 2010). Several processing steps, such as skull stripping, Talairach transforms, atlas registration, as well as spherical
surface maps and parcellations are then initialized using common
information from the within-subject template. This analysis method
has been validated and shown to signicantly increase reliability and
statistical power (Reuter et al., 2012).
2.5. Statistical analysis
2.5.1. Cross-sectional analyses
ManneWhitney U and Fisher exact tests were used to compare
demographic and clinical data. Cross-sectional volume differences
were compared between ALS and healthy controls using an analysis
of covariance (ANCOVA). All of these analyses were adjusted for age
and gender. Because of possible non-normality, we applied permutation tests with 10,000 permutations according to participant
groups.
2.5.2. Relationship between imaging and clinical characteristics
The relationship between cross-sectional clinical data and
volumes in ALS patients was assessed using a combination of an
ANCOVA and principal component analysis (PCA). PCA is a
method that is used to describe a large group of variables by a
smaller group of principal components (PCs), each consisting of a
number of interrelated variables. Of all subcortical structures (n
20), 3 PCs were retained (Supplementary Fig. 1). These PCs were
linearly regressed using clinical data (using ANCOVA) to assess the
relationship between clinical data and volumes (represented by
PCs), thereby adjusting for age and gender. This method was used
because it takes advantage of the underlying correlation of
different subcortical structures (e.g., the ventricles are actually
part of 1 system; see also Supplementary Fig. 1) and increase both
power and robustness by grouping variables that share common
features (in PCs), reducing the number of comparisons, and
minimizing possible effects of multicollinearity. The relationship
between the PCs of the subcortical structures and survival was
studied using univariate and multivariate Cox proportional hazards models. Known independent predictors of survival (age at
onset and bulbar onset) were included in the multivariate analysis. Gender was not statistically signicantly associated with
survival and was therefore excluded from the multivariate survival analysis.
2.5.3. Longitudinal analyses
A linear mixed-effects model (LME) was used to assess the rate
of change of subcortical volumes in ALS patients over time while
accounting for random between-subject variation (Bernal-Rusiel

H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082


Table 1
Demographic and clinical characteristics of study subjectsa
ALS patients

Age
Gender (male:female)
Handedness (left:right)c
ALSFRS-R score
Site of onset (%)
Bulbar
Cervical
Lumbosacral
Disease progression rate
(points decrease/month)d
El Escorial criteria (%)
Denite
Probable
Probable laboratory
supported
Possible
Disease duration (months)
Type (sporadic:familial ALS)
C9orf repeat expansion
Died

Healthy
controls

Baseline
(n 112)

Follow-up
(n 39)

Baseline
(n 60)

60.4 (24e78)
86:26
18:86
41 (29e48)

59.9 (24e78)b 60.4 (29e76)


30:9
46:14
5:32
7:44
36 (25e46)

24 (21)
55 (49)
33 (30)
0.5 (02.6)e

3 (8)
24 (62)
12 (31)
0.6 (0.5 to 3)f

13 (12)
48 (43)
35 (31)

4 (10)b
19 (49)b
10 (26)b

16 (14)
14.2 (4e75)
105:7
7/112
77/112

6 (15)b
18.5 (10e82)
37:2
5/39
23/39

0/60

Key: ALSFRS-R, revised ALS Functional Rating Scale.


a
Values are in median (range) unless otherwise specied.
b
At baseline.
c
Missing data for ALS patients (n 8), follow-up ALS patients (n 2), healthy
controls (n 9).
d
Points decrease are points decrease on the ALSFRS-R score.
e
Disease progression rate for baseline was calculated using the formula (48 
ALSFRS-R score)/disease duration (in months).
f
Disease progression rate for follow-up was calculated using the formula
(ALSFRS-R score on baselineeALSFRS-R score on follow-up)/time since baseline MRI
(in months).

et al., 2012). Age and gender were included as covariates in this


analysis.
All tests were 2-tailed, and p-values <0.05 were considered to be
statistically signicant. A false discovery rate (FDR) correction for
multiple testing was performed. Data was reported as mean 
standard error of volume in mm3 (unless otherwise specied).
Statistical analyses were performed using the software program R
(http://cran.r-project.org).
3. Results
3.1. Clinical characteristics
A total of 112 ALS patients and 60 healthy controls were enrolled in
this study (Table 1). Patients and controls were well matched for age
(p 0.414), gender (p 1.000), and handedness (p 0.648). The
median ALSFRS-R score was relatively high (41) because of the relatively short median disease duration (14.2 months; Supplementary
Fig. 3 for distribution of the disease duration). Seven (6.3%) ALS patients carried the C9orf72 repeat expansion, and 77 (68.8%) ALS patients died. Although there was a trend for longer disease duration in
familial ALS patients (median [range]: 21.16 [5e75]) compared to
sporadic patients (13.14 [4e59]), this was not statistically signicant
(p 0.057).
3.2. Cross-sectional analysis
The outcomes of the volumetric analysis of the DGM, ventricles,
and hippocampal subelds are shown in Table 2. Intracranial volumes in ALS patients and healthy subjects were similar (p 0.672).
The cross-sectional analysis revealed that patients with ALS

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compared to healthy controls had the following: 1) signicantly


lower hippocampal volumes (left: 195  71 mm3, p 0.007;
right 173  67 mm3, p 0.011); 2) larger inferior lateral ventricles
(left: 186  76 mm3, p 0.013; right: 114  56 mm3, p 0.041); and
3) a smaller left presubiculum (29  11 mm3; p 0.009). Trends
for volume differences were found for the right presubiculum (p
0.052) and for the subiculum (left: p 0.091; right: p 0.062). The
volume of CA1 was similar in patients and healthy controls. We
additionally studied the relationship between age and hippocampal
volume as shown in Supplementary Fig. 2. This gure shows that
the hippocampal volume is smaller in ALS patients compared with
healthy subjects, irrespective of age.
In addition, the shape of the thalamus, putamen, and caudate
nucleus and nucleus accumbens was analyzed. As shown in Fig. 1, 1
cluster of regional volume change was found in both thalami (left:
p 0.008; right: p0.026). Based on anatomical knowledge, this
cluster would appear to be located at the point where the intramedullary lamina (IML) enters and exits the thalamus (Zarei et al.,
2010). The shape analysis of the putamen, caudate nucleus, and
nucleus accumbens did not reveal differences between ALS patients
and healthy controls. Comparison of sporadic versus familial ALS
patients did not reveal any statistically signicant differences.

Table 2
Age- and gender-adjusted subcortical volume differences in study subjectsa

Left
Thalamus
Caudatus
Putamen
Pallidum
Hippocampus
Amygdala
Accumbens
Right
Thalamus
Caudatus
Putamen
Pallidum
Hippocampus
Amygdala
Accumbens
Ventricles
Left lateral
Left inferior lateral
Right lateral
Right inferior lateral
Third
Fourth
Hippocampal subelds
Left
CA1
CA2/3
CA4/DG
Presubiculum
Subiculum
Right
CA1
CA2/3
CA4/DG
Presubiculum
Subiculum

Healthy controls ALS patients

Difference

p Valueb

6621  83
3673  54
5562  67
1678  26
3892  58
1615  31
577  15

6475  61
3642  39
5497  49
1655  19
3697  42
1572  23
545  11

146  103
32  67
65  83
23  32
195  71
43  39
32  19

0.159
0.636
0.435
0.468
0.007**
0.274
0.091

6474  83
3730  55
5326  70
1575  21
4034  54
1831  35
688  13

6304  60
3645  40
5325  51
1528  16
3861  40
1816  25
678  10

170  102
85  69
2  87
47  26
173  67
16  43
10  16

0.098
0.216
0.984
0.078
0.011*
0.719
0.561

13,337  1036
556  61
11,502  817
501  45
1481  74
1986  90

14,766  757
742  45
12,951  598
615  33
1617  54
2162  66

1429  1284
186  76
1449  1014
114  56
136  92
176  111

0.273
0.013*
0.155
0.041*
0.144
0.117

4  6
28  20
14  11
29  11
23  13

0.509
0.162
0.209
0.009**
0.091

5  6
15  18
10  10
21  11
24  13

0.417
0.409
0.329
0.052
0.062

335
972
549
491
646







5
16
9
9
11

346  5
1027  15
572  8
473  9
643  10

331
944
535
462
624







4
12
7
6
8

341  3
1012  11
562  6
452  6
618  8

Hippocampal atrophy is present on both sides and enlarged inferior lateral ventricles. Atrophy of the left presubiculum was statistically signicant; on the right side
there was a tendency to atrophy in ALS patients (p 0.052). Values are in mean 
standard error.
Key: ALS, ALS patients; CA, cornu ammonis; CO, healthy controls; DG, dentate gyrus.
* p < 0.05
** p < 0.01.
a
Volumes are reported in mm3.
b
p Values were completely randomly permutated 10,000 times.

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H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082

Fig. 1. Thalamic shape. Comparison of thalamic shape of ALS patients with healthy subjects. The orange areas indicate affected aspects. These areas correspond closely to the
intramedullary laminae of the thalamus. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

3.3. Relationship with clinical characteristics


Principal component analysis is a method that allows different
variables to be grouped into a smaller number of representative
variables (Supplementary Fig. 1). As shown in this gure, the rst PC
represents the basal ganglia and accumbens areas on both sides;
the second PC represents the ventricles; and the third PC represents
the limbic structures of the DGM (both hippocampi and amygdalae). The results of the regression of these PCs with clinical
characteristics are listed in Table 3. In summary, larger ventricles
were signicantly associated with a lower ALSFRS-R score (1.06 
0.47, p 0.026). In addition, smaller basal ganglia, smaller limbic
structures, and larger ventricles were associated with a shorter
survival (basal ganglia: hazard ratio [HR] 1.44 [95% condence
interval: 1.13e1.82), p 0.003; ventricles: HR 1.28 [1.03e1.59],
p 0.029; limbic structures: HR 1.31 [1.08e1.60], p 0.007).
With adjustment for bulbar onset, effects remained statistically
signicant for basal ganglia and limbic structures, but with additional adjustment for age at onset, components did not remain
statistically signicant.
3.4. Longitudinal analysis
This analysis showed progressive enlargement of the lateral
ventricles (left: 1090.8  223.3, p < 0.001; right: 964.7  208.9, p <
0.001), the right inferior lateral ventricle (60.1  24.6, p 0.014)
and the third and fourth ventricles (third: 69.7  18.2, p < 0.001;

fourth: 105.5  31.8, p 0.001) in ALS patients. DGM volume did


not change signicantly during follow-up in ALS patients. Figure 2
and Supplementary Table 1 summarize the results of the longitudinal analysis of subcortical structures. They also show that volume
decrease of the hippocampal subelds was progressive in the left
presubiculum (22.8  6.3 mm3; p < 0.001), the right CA2/3 (22.7
 7.5, p 0.002), and CA4/DG (8.7  4.4; p 0.045). Effects of
volume increase of the lateral ventricles did reach FDR correction
for multiple testing (q 0.05), other comparisons did not.
4. Discussion
ALS patients show a pattern of subcortical involvement characterized by hippocampal and thalamic atrophy, as well as ventricular
enlargement. Hippocampal involvement is most severe and progressive in the left presubiculum, and this is accompanied by
enlarged temporal ventricles. During follow-up, we also observed
shrinkage in the right CA2/3 and CA4/DG, as well as enlarging ventricles (both lateral, right inferior lateral, third and fourth ventricles),
indicating cerebral disease progression. Although the thalamus volume was not signicantly different in patients compared to controls,
shape analysis was suggestive of atrophy of the thalamic intramedullary lamina (IML) that interacts with frontostriatal circuits
(Zarei et al., 2010). With respect to clinical measures, we found that
larger ventricular volume at baseline correlated with lower ALSFRS-R
scores. Considering the differences found between ALS patients and
healthy subjects and the associations with clinical measurements, it

H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082

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Table 3
Correlations between subcortical structures and clinical parameters in study subjects

Progression rate (univariate)a


Progression rate (multivariate)a,b
ALSFRS-R (univariate)
ALSFRS-R (multivariate)b
Survival (univariate)c
Survival (multivariate)c,d

PC1 (basal ganglia), mean  SE; p value

PC2 (ventricles), mean  SE; p value

PC3 (limbic structures), mean  SE; p value

0.04  0.04; 0.316


0.01  0.06; 0.814
0.85  0.43; 0.049*
0.73  0.55; 0.185
1.44 (1.13e1.82); 0.003**
1.12 (0.85e1.48); 0.410

0.06  0.04; 0.145


0.09  0.05; 0.060
0.71  0.43; 0.100
1.06  0.47; 0.026*
1.28 (1.03e1.59); 0.029*
1.03 (0.79e1.33); 0.844

0.03  0.04; 0.524


0.01  0.05; 0.783
0.40  0.43; 0.351
0.17  0.50; 0.736
1.31 (1.08e1.60); 0.007**
1.05 (0.81e1.35); 0.719

At baseline, higher ventricular volume is associated with lower ALSFRS-R score. Survival is shorter in patients with smaller basal ganglia, larger ventricular volume, and smaller
limbic structures (see also Fig. 2).
Key: ALSFRS-R, revised ALS Functional Rating Scale; PC1, principal component 1 (representing basal ganglia); PC2, principal component 2 (representing ventricles); PC3,
principal component 3 (representing both hippocampi and amygdalas).
* p < 0.05.
** p < 0.01.
a
Progression rate (48  ALSFRS-R score)/disease duration (in months).
b
Covariates were age and gender.
c
Values are hazard ratio (95% condence interval).
d
Covariates were age at onset and bulbar onset.

is likely that subcortical structures play a role in the neurodegenerative process of ALS. In addition, shorter survival was related to
smaller basal ganglia and limbic structures and larger ventricles, but
multivariate analyses showed that age at onset was associated more
strongly with survival than the above-mentioned PCs.
Only 1 study reported a detailed cross-sectional analysis of the
deep gray matter in 39 ALS patients (Bede et al., 2013). Hippocampal atrophy at baseline was comparable in these studies, suggesting a role of the hippocampus in the neurodegenerative process
and clinical phenotype of ALS patients. The shape analysis of the
thalami was largely comparable in this study and in our examination (Bede et al., 2013). In our study in 112 ALS patients, however, a
change in thalamic shape was not observed to be accompanied by
volume changes of the entire thalamus. Neither shape nor
morphometric analysis showed (regional) atrophy of other DGM,
whereas the above-mentioned study did report atrophy of the
caudate nucleus, accumbens area, and putamen. A possible explanation for this difference is the shorter disease duration in our
study (14 vs. 26 months). A subgroup analysis (n 26) was performed (mean disease duration  standard deviation: 24.3 
10.4 months) to further elucidate this difference. With respect to
the DGM, the right hippocampus (p 0.028) showed a signicant
difference in this specic subgroup. This comparison is, however,
hampered because of the relatively small sample size and different
distribution of the data, and because other demographic characteristics such as ALSFRS-R score and disease progression were not
available for comparison. Although our longitudinal analysis
showed a tendency for the putamen, caudate nucleus, and right
accumbens area to decrease in volume, these decreases did not
reach statistical signicance (Fig. 2). This rst longitudinal analysis
of subcortical structures also showed progressive ventricular
enlargement and decreasing volumes of some hippocampal subelds, indicating progressive neurodegeneration. Hippocampal
subelds have not previously been studied in vivo, but the nding
of hippocampal subeld degeneration is supported by post mortem
histological research (Takeda et al., 2009; Zu et al., 2013), and the
present study shows that it can also be detected in vivo at a relatively early stage of the disease.
Ventricular enlargement at baseline was restricted to the temporal parts of the ventricular system (in line with hippocampal
atrophy) and progressed during follow-up to signicant enlargement of both lateral ventricles and third and fourth ventricles.
These results suggest that neurodegeneration in the temporal lobe
is an early characteristic of ALS and is present before progressive
neurodegeneration becomes visible in ventricular enlargement in
the rest of the brain. Ventricular enlargement is a common feature

of neurodegenerative diseases but has not previously been studied


in ALS (Thompson et al., 2004). This study showed that larger
ventricular volume in ALS is also correlated with a lower functional
motor score (ALSFRS-R) and shorter survival. In addition, smaller
basal ganglia and smaller limbic structures at baseline were associated with shorter survival. Multivariate survival analyses, however, showed age at onset to be associated more strongly with
survival than the PCs of basal ganglia, ventricles, and limbic structures. The association between age of onset and survival was not
studied further because this has been studied in detail before (Chio
et al., 2009). Analysis of individual brain structures (rather than
PCs) in larger cohorts might provide more insight into the relationship between individual structures and survival.
Although hippocampal atrophy is a nonspecic feature of
various brain disorders, the pattern of hippocampal subeld
degeneration might be more specic for different diseases and
might be related to the cognitive prole of ALS patients (Frisoni
et al., 2008; Lindberg et al., 2012). For example, it is suggested
that the presubiculum is involved in processing spatial information,
which is in line with the decits on spatial working memory tasks
observed in ALS patients (Hammer et al., 2011; Jarrard et al., 2004).
With respect to the other hippocampal subelds, it is important to
note that no volume change of the CA1 was observed at baseline or
during follow-up, which is in accordance with clinical observations.
Clinically, involvement of the CA1 causes severe amnesia, known
from diseases such as Alzheimers disease and transient global
amnesia (Bartsch et al., 2006). Although memory impairment is
reported to be present in ALS, severe amnesia is atypical for ALS in
the early stages. Histopathological studies have reported involvement of the CA1, but this might develop later in the disease and is
not always accompanied by neuronal cell loss, thereby possibly
explaining why no atrophy of the CA1 was found (Brettschneider
et al., 2013; Takeda et al., 2009). Detailed interpretation of the
clinical signicance of these ndings requires studies that combine
extensive cognitive measurements and neuroimaging data, and
these are currently scarce.
The pattern of regional atrophy of the thalamus described here is
similar to the results of a recent study (Bede et al., 2013). Although
not described as the thalamic IML by Bede et al., based on the
anatomy of the thalamus, these clusters correspond to the thalamic
IML. Additional atrophy of the anterior and anterodorsal nucleus
might contribute to this change, but cannot explain the entire
pattern (Zarei et al., 2010). This change was quite subtle, as it did not
signicantly affect the whole thalamic volume, but it might be
clinically signicant because of its inuence on frontal cognitive
functions and, for example, because it correlates with verbal uency

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H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082

Fig. 2. Longitudinal analysis of subcortical structures. The x-axis shows the number of the magnetic resonance imaging (MRI) scan (1 rst scan; 2 second scan). The y-axis
shows the volumes normalized to healthy subjects, meaning that 100% was the mean subcortical volume of healthy subjects at baseline. This gure shows signicantly smaller
volumes of both hippocampi and signicantly larger volumes of both inferior lateral ventricles in ALS patients at baseline. After a follow-up of 5.5 months (on average), the volumes
of the right CA2/3 and CA4/DG decreased signicantly. In addition, volumes of nearly all ventricles increased signicantly during follow-up. The colors in the graphs correspond to
the colors in the pictures of subcortical structures and hippocampal subelds. Error bars indicate standard errors. Abbreviations: CA, cornu ammonis; DG, dentate gyrus; Sub,
subiculum; Presub, presubiculum. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this
article.)

(Van der Werf et al., 2000; Zarei et al., 2010). Surprisingly, no shape
alteration was observed in the nucleus connected with the motor
cortex, the ventral lateral nucleus.
Although a relatively large number of patients and controls were
studied in detail, several limitations of this study should be taken
into account. First, no follow-up of healthy controls was performed.
Normal aging has, however, been extensively studied, and shows

that DGM volume usually decreases by less than 0.5% and ventricle
size increases by less than 3% in 6 months in healthy subjects (Fjell
et al., 2009). It is therefore highly unlikely that the volume changes
in this study are due to normal aging (e.g., interior lateral ventricles
of ALS patients increased to 130%e140% the size of normal subjects
in 6 months) (Fjell et al., 2009). Second, males were somewhat
overrepresented in this study, based on what is known from

H.-J. Westeneng et al. / Neurobiology of Aging 36 (2015) 1075e1082

population-based studies; but gender was well matched between


ALS patients and healthy subjects (Huisman et al., 2011). Third,
more detailed neuropsychological examination and correlation
with, for example, hippocampal atrophy would have been interesting (Bede et al., 2013). With respect to the outcomes of the
hippocampal subeld analysis, assessment of spatial working
memory function would have been of interest. Larger studies
combining neuropsychological and histopathological data with
structural and functional MRI data are therefore of great importance. The NeuroImaging Society in ALS (NISALS) might provide an
opportunity to realize this (Turner et al., 2011).
5. Conclusions
The present study shows that ALS patients have reduced hippocampal volumes at an early stage of the disease, the area most
affected being the presubiculum. This, combined with ventricular
enlargement, found to be progressive during follow-up, was associated with survival and ALSFRS-R. Furthermore, thalamic degeneration was found to be most probably located in the IML. In
conclusion, subcortical involvement is progressive and correlates
with clinical parameters in ALS, underscoring its role in the pathophysiology of ALS.
Disclosure statement
Henk-Jan Westeneng has nothing to disclose.
Esther Verstraete has nothing to disclose.
Rene Walhout has nothing to disclose.
Jeroen Hendrikse has nothing to disclose.
Jan H. Veldink has nothing to disclose.
Martijn P. van den Heuvel has nothing to disclose.
Leonard H. van den Berg reports grants from Netherlands ALS
Foundation, grants from Prinses Beatrix Spierfonds, grants from
Netherlands Organisation for Health Research and Development (Vici
scheme), grants from European Communitys Health Seventh
Framework Programme (FP7/2007e2013) (grant agreement no.
259867), during the conduct of the study; personal fees from Baxter
for Scientic Advisory Board and Travel Grant, and personal fees from
Scientic Advisory Board BiogenIdec, outside the submitted work.
Acknowledgements
This work was supported by the Netherlands ALS Foundation,
Prinses Beatrix Fonds, Netherlands Organization for Health
Research and Development (Vici scheme to LHvdB), the Nederlandse Organisatie voor Wetenschappelijk Onderzoek under the
frame of E-RARE-2, the ERA-Net for Research on Rare Diseases, and
the European Communitys Health Seventh Framework Programme
(FP7/2007e2013) under grant agreement (259867).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version at http://dx.doi.org/10.1016/j.neurobiolaging.
2014.09.002.
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