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RESEARCH ARTICLE
Grey Matter Microstructural Integrity
Alterations in Blepharospasm Are Partially
Reversed by Botulinum Neurotoxin Therapy
Hanganu Alexandru
1☯
, Muthuraman Muthuraman
1,2☯
, Venkata Chaitanya Chirumamilla
2
,
Nabin Koirala
2
, Burcu Paktas
1
, Gu
¨nther Deuschl
1
, Kirsten E. Zeuner
1
, Sergiu Groppa
1,2
*
1Department of Neurology, University of Kiel, Kiel, Germany, 2Movement Disorders and Neurostimulation,
Department of Neurology, Neuroimage Center (NIC) of the Focus Program Translational Neuroscience
(FTN), University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
☯These authors contributed equally to this work.
*segroppa@uni-mainz.de
Abstract
Objective
Benign Essential Blepharospasm (BEB) and hemifacial spasm (HFS) are the most common
hyperkinetic movement disorders of facial muscles. Although similar in clinical presentation
different pathophysiological mechanisms are assumed. Botulinum Neurotoxin (BoNT) is a
standard evidence-based treatment for both conditions. In this study we aimed to assess
grey matter microstructural differences between these two groups of patients and compared
them with healthy controls. In patients we furthermore tracked the longitudinal morphometric
changes associated with BoNT therapy. We hypothesized microstructural differences
between the groups at the time point of maximum symptoms representation and distinct lon-
gitudinal grey matter dynamics with symptom improvement.
Methods
Cross-sectional and longitudinal analyses of 3T 3D-T1 MRI images from BEB, HFS patients
prior to and one month after BoNT therapy and from a group of age and sex matched
healthy controls. Cortical thickness as extracted from Freesurfer was assessed as parame-
ter of microstructural integrity.
Results
BoNT therapy markedly improved motor symptoms in patients with BEB and HFS. Signifi-
cant differences of grey matter integrity have been found between the two patients groups.
The BEB group showed lower cortical thickness at baseline in the frontal-rostral, supramar-
ginal and temporal regions compared to patients with HFS. In this group BoNT treatment
was associated with a cortical thinning in the primary motor cortex and the pre-supplemen-
tary motor area (pre-SMA). Contrary patients with HFS showed no longitudinal CT changes.
A decreased cortical thickness was attested bilaterally in the temporal poles and in the right
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 1 / 9
a11111
OPEN ACCESS
Citation: Alexandru H, Muthuraman M,
Chirumamilla VC, Koirala N, Paktas B, Deuschl G,
et al. (2016) Grey Matter Microstructural Integrity
Alterations in Blepharospasm Are Partially
Reversed by Botulinum Neurotoxin Therapy. PLoS
ONE 11(12): e0168652. doi:10.1371/journal.
pone.0168652
Editor: Lutz Jaencke, University of Zurich,
SWITZERLAND
Received: April 20, 2016
Accepted: December 4, 2016
Published: December 16, 2016
Copyright: ©2016 Alexandru et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: The data used in this
study are from patients so we are not allowed to
upload the data to any public repository. However
we can distribute the data for research purposes in
an anonymous form. On behalf the authors we give
the confirmation that the data will be available upon
request to all interested researchers. [Prof. Dr.-
Ing. M.Muthuraman, Biomedical statistics and
multimodal signal processing unit, Movement
Disorders and Neurostimulation, Department of
Neurology, Focus Program Translational
superior frontal region in BEB patients in comparison to HC. Patients in the HFS group pre-
sented a decreased CT in the left lingual gyrus and temporal pole.
Conclusions
Although patients with BEB and HFS present clinically with involuntary movements of facial
muscles, they exhibited differences in cortical thickness. While BoNT therapy was equally
effective in both groups, widespread changes of cortical morphology occurred only in BEB
patients. We demonstrated specific disease- and therapy-dependent structural changes
induced by BoNT in the studied hyperkinetic conditions.
Introduction
Benign Essential Blepharospasm (BEB) and Hemifacial Spasm (HFS) are the most common
movement disorders affecting the face. BEB is a bilateral condition characterized involuntary
closure of the eyelids caused by spasms of the orbicularis oculi muscle[1]. In contrast, HFS is
characterized by unilateral, intermittent muscular contractions of the eye or facial muscles [2].
Both conditions are clinically similar, but BEB is considered to result from a dysfunction of the
basal-ganglia-cortical loops with an involvement of the sensorimotor cortical regions [3,4],
while muscular contractions in HFS are caused often by vessel compression or irritation of the
facial nerve [5]. Botulinum neurotoxin (BoNT) therapy is the standard symptomatic treatment
for both conditions [6].
The exact pathophysiological changes leading to BEB are not completely clear. Sensory dis-
crimination is disturbed in BEB patients [7,8] and processing of tactile stimuli is impaired due
to abnormal sensorimotor integration [9]. In patients with BEB grey matter increases in the
bilateral putamen were detected in a structural neuroimaging study [10]. However, this finding
was contrasted by another voxel based morphometry study that detected grey matter intensity
increase in the caudate and cerebellum bilaterally, combined with a decrease in the putamen
and thalamus bilaterally [11]. In a cross sectional study between BEB and healthy controls,
grey matter increases were seen in the right middle frontal gyrus, while lower grey matter vol-
ume was detected in the left post central and left superior temporal gyrus [12]. Functional
cross sectional studies on BEB and cervical dystonia have shown increased basal ganglia activa-
tion with motor tasks [13]. However, these functional and structural changes demonstrated in
patients with focal or generalized dystonia in comparison to healthy controls cannot be clearly
classified as primary or secondary. Similarly there is no clear data on cerebral morphological
changes in patients with HFS. The continuous muscular activity might directly or indirectly
modify the cortical sensory-motor network, leading to secondary structural changes. To
improve our understanding of dystonic conditions an exact characterization of primary struc-
tural integrity changes are necessary. Continuous tonic or phasic movements induce at short
intervals secondary functional and structural alterations of the involved cerebral networks that
can be then barely distinguished from elemental pathophysiological fingerprints of the disease.
The purpose of our study was to analyze the cortical thickness in BEB compared to HFS
patients. Both conditions present with clinically similar symptoms, but exhibit different patho-
physiological backgrounds. In the proposed analysis of both groups a clear differentiation of
primary and secondary cause of these morphological changes can be estimated. Moreover we
postulate that the primary and secondary structurally modified networks respond to the
changed sensorimotor input with immobilization after BoNT in a different way. Therefore the
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 2 / 9
Neuroscience (FTN), Johannes-Gutenberg-
University Hospital, Langenbeckstr. 1, 55131
Mainz, Germany, Email: mmuthura@uni-mainz.de.
Funding: This work was supported by the German
Research Council (CRC 1193, project B05). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
selected group of HFS patients was recruited as controls with a very similar clinical presenta-
tion for BEB patients. We contrast BEB and HFS patients with healthy controls in order to
improve comparability and track the primary and secondary functional and morphometric
changes by chronic muscular hyperactivity.
Materials and Methods
Patients
13 patients with BEB and 11 patients with HFS were included in this study. The demographic
details are given in Table 1. Participants were studied twice: 1. at the time point of maximal
symptomatic phase and 4 weeks later. A clinical examination including Unified Dystonia Rat-
ing Scale (UDRS), Blepharospasm Rating Scale (BRS),[14], Blepharospasm Disability Scale
(BDS) and the Severity Rating Scale (SRS) was performed [15]. During the first session each
patient was treated with intramuscular BoNT. The intramuscular injection was performed by
clinicians with expertise and specialized training in BoNT administration, who were blinded
to the aim of this study. BEB and HFS diagnosis was based on previously published criteria
[16]. Additionally, as a control experiment we did a cross sectional analyses between the two
patient groups at time 1 and 20 age and sex matched healthy controls. The demographics of
the healthy controls are given in Table 1.
Clinical evaluation
The UDRS comprised ratings for 14 body areas including eyes and upper face, lower face, jaw
and tongue, larynx, neck, trunk, shoulder/proximal arm, distal arm/hand, proximal leg, distal
leg/foot. For each of the 14 body areas assessed, the UDRS quantified a severity and a duration
score. The severity scale ranged from 0 (no dystonia) to 4 (extreme dystonia). The duration
scale measured whether dystonia occurred at rest or with action, and the scale ranged from 0 to
4. Both, the severity and the duration score summarized to the total UDRS score. We applied
the BRS to evaluate the location, influencing factors, severity of involuntary movements and
disability. Higher BRS scores indicated increased disability due to BEB. With the BDS we quan-
tified additional impairments in everyday life, and we assessed 8 domains at a scale from 1
(uncomfortable, but no limitation) to 5 points (marked limitation due to BEB). The treatment
efficacy of BoNT was rated by ‘relief’ of ‘improvement’ of symptoms without specifying the
nature of the improvement. We adopted the SRS to determine the severity of BEB on a scale
from 0 (no BEB present) to 4 (severe, forceful contractions). In all participants we excluded any
history of neurologic or psychiatric disorders as well as antidepressive, neuroleptic or sedative
medications. The clinical evaluation is summarized in Table 2. Paired samples t-tests were used
for the between groups comparison. All participants gave written informed consent before the
Table 1. Demographic description of groups.
BEB HFS HC P
1
P
2
P
3
Age 65.04±6.21 58.62±11.18 57.52±9.11 0.089 0.074 0.86
Gender 4/9 4/7 10/10 0.92 0.24 0.12
BoNT_ R, U 37.69±27.20 32.73±37.37 - 0.71 - -
BoNT_L, U 36.92±27.58 25.00±36.61 - 0.37 - -
Abbreviations:HC = Healthy controls; BoNT = botulinum neurotoxinR/L = right side/ left side treatment was applied to the right/left side of face; U = units;
p = p-value,significance of differences; P
1
–Comparison between BEB and HFS; P
2
–Comparison between BEB and HC; P
3
–Comparison between HFS
and HC;
doi:10.1371/journal.pone.0168652.t001
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 3 / 9
study. The study was conducted in full accordance to the Declaration of Helsinki and had been
approved by the local ethics committee in Medical faculty University clinic, Kiel.
Image acquisition and analyses
Images were acquired at a 3 Tesla MRI scanner (Achieva; Philips, Best, the Netherlands)
equipped with an 8-channel head coil. A T1-weighted echo-planar imaging sequence (1 mm
slice thickness, 208 ×208 matrix, TE = 3.6 ms, TR = 7.8 ms, flip angle = 8 degrees) was used.
Cortical reconstruction and volumetric segmentation was performed with FreeSurfer 5.3 image
analysis suite (Massachusetts General Hospital, Harvard Medical School; http://surfer.nmr.mgh.
harvard.edu). Briefly, this included motion correction and averaging of multiple volumetric T1
weighted images, removal of non-brain tissue using a hybrid surface deformation procedure
[17], automated Talairach transformation, segmentation of the subcortical white matter and
deep gray matter volumetric structures[18], intensity normalization[19], tessellation of the gray/
white matter boundary, automated topology correction[20], and surface deformation following
intensity gradients to optimally place the gray/white and gray/cerebrospinal fluid borders[21].
Images were automatically processed with the longitudinal module included in Freesurfer
[22]. The exact procedure is described elsewhere [23].
For the analysis, we applied a general linear model of statistical analysis to establish the
cross-sectional differences between the groups. Further we quantified the longitudinal changes
by computing the rate of change of cortical thickness (mm/year) between the groups using the
formula: (thickness at Time 2 –thickness at Time 1) / (Time 2 –Time 1). Cortical thickness was
smoothed with a 10-mm Gaussian kernel to reduce local variations in the measurements [24].
Statistical differences were computed using a random effects model with t-tests for each corti-
cal location. For statistical difference maps the significant threshold was set to an uncorrected
p-value of 0.001 (two-tailed). Further, correction using the Monte-Carlo based on the simu-
lation (cluster analyses) adjustment was applied with a p-value of 0.05. Finally, we estimated
the Pearson correlation (corrected for multiple comparisons using Bonferroni correction)
between the change in clinical scores and the change in cortical thickness for both the BEB and
HFS group separately for each hemisphere.
Results
There were no significant differences between the groups in respect to demographics, adminis-
tered dosage UDRS, BRS and SRS scale (Table 2). BoNT treatment improved the dystonic
Table 2. Clinical scores from both groups.
BEB HFS
UDRS at t1 4.27±2.37 4.64±2.61
UDRS at t2 2.69±1.38 3.27±2.21
BRS at t1 8.85±2.38 9.82±3.66
BRS at t2 6.69±2.95 8.09±3.73
BDS at t1 10.38±2.63 7.73±0.65
BDS at t2 9.46±2.18 7.45±0.52
SRS at t1 2.23±0.83 2.09±0.70
SRS at t2 1.46±0.66 1.09±0.83
Abbreviations: UDRS = Unified Dystonia Rating Scale; BRS = Blepharospasm Rating Scale;
BDS = Blepharospasmus Disability Scale; SRS = Severity Rating Scale; t1 = time 1; t2 = time 2;
doi:10.1371/journal.pone.0168652.t002
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 4 / 9
contractions in BEB patients, as measured with the UDRS and SRS scales (Table 2). HFS
patients improved as well at time 2 on the SRS scale. The groups differed in the level of disabil-
ity (BD scale) at baseline as well as at time 2 after BoNT treatment.
We performed cross-sectional analyses between the BEB at time 1 and the healthy controls,
and found that cortical thinning occurred in BEB at the left temporal pole, right lateral occipi-
tal, right superior frontal and right inferior temporal regions. Similarly, in the comparison of
HFS patients to HC, we found a cortical thinning in subjects with HFS at the left lingual and
left posterior bank of transversal temporal gyrus. The complete description of the results with
t-values, voxel size and Talairach coordinates are given in Tables 3and 4.
The analysis of cortical thickness has been performed in two stages. First, we performed an
intergroup cross-sectional analysis at time 1, i.e. BEB vs. HFS at time 1 and an additional anal-
ysis at time 2 (Fig 1). In comparison to HFS, the BEB group at time 1 revealed differences in
cortical thickness in the primary motor cortex bilaterally, left inferior temporal, right middle
temporal, right frontal-rostral and right supramarginal regions. After BoNT-treatment, corti-
cal thickness changed in the primary motor cortex and in the left pre-SMA of the BEB group
compared to HFS.
The analysis for each group individually revealed no cortical thickness changes over time.
The differential analysis of the longitudinal change between the groups showed a higher rate of
cortical thinning in BEB patients over time in the left pre-SMA and the right insula in compar-
ison to HFS subjects. From the correlation analyses of change in clinical parameters to the
change in cortical thickness we found the change in cortical thickness in BSF patients over
time (t0-t1) was significantly (p = 0.0105) correlated (r = -0.6804) to change (t0-t1) in BRS
only for the right hemisphere. No other significant correlations were found for any other clini-
cal parameters or for the HFS group.
Discussion
Patients with BEB exhibited bilateral increased cortical thickness in the sensorimotor cortex
compared to HFS. Cortical thickness decreased in BEB patients after BoNT treatment while no
changes were noted in the HFS group. This finding might be attributed to cortical reorganiza-
tion over time after BoNT treatment. The exact functional and structural changes that occur
with improvement of symptoms in patients with dystonic or hyperkinetic disorders after
BoNT therapy are not clear. Previous studies demonstrated that long term potentiation
Table 3. Statistical significant clusters for the group comparison between patients with BEB and HC.
Right Hemisphere
Cluster No t-Max Size (mm
2
) Tal X Tal Y Tal Z Annotation
1-4.5331 116.32 26.7 -97.7 -3.6 Lateral Occipital
2-4.0909 95.44 7.0 -0.6 64.6 Superior Frontal
3-3.6567 29.55 46.6 -5.6 -33.2 Inferior Temporal
Left Hemisphere
1-4.0565 124.91 -34.9 5.2 -30.7 Temporal pole
doi:10.1371/journal.pone.0168652.t003
Table 4. Statistical significant clusters for the group comparison between patients with HFS and HC.
Left Hemisphere
1-4.0565 52.14 -13.6 -54.0 -2.9 Lingual
2-4.0414 21.11 -55.1 -42.6 -4.0 Posterior part of the tranverse temporal gyrus
doi:10.1371/journal.pone.0168652.t004
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 5 / 9
(LTP)–like plasticity studied using transcranial magnetic stimulation was abnormal in BEB
patients, but could be restored after BoNT treatment [25]. Possibly these functional changes
are reflected in our study by the decreased cortical thickness in the sensorimotor cortex after
the BoNT in BEB patients. Systematically the somatotopic representations of punctate tactile
stimuli were mapped before and after BoNT therapy and showed deficient activation in pri-
mary and secondary somatosensory representations and described modulation of basal ganglia
activation might reflect an indirect effect of the BoNT [26]. The longitudinal structural adapta-
tion in our study was specific for BEB patients pointing out a disease specific reorganization.
The morphological differences involved the motor system, but were also accompanied by
changes in the secondary and tertiary associative cortices.
Previous studies suggested that BEB pathophysiology is associated with changes in the pri-
mary motor cortex [27]. Specifically, gray matter reductions as shown by voxel-based mor-
phometry (VBM) in the facial portion of the primary motor cortex have been described in
BEB when compared to healthy controls [28]. This result was not replicated by our data. The
significant cluster in the comparison of BEB patients with HC was more rostral and could be
related to M1 interconnected areas in the frontal cortex. For the main hypothesis of our study
we address however the contrast of BEB and HFS patients to exclude unspecific and secondary
changes. A further great advantage of our analysis is the use of cortical thickness measure-
ments and not VBM-related intensity analysis, which might represent a less sensitive measure
of grey matter integrity. Therefore, our analysis showed higher cortical thickness values in the
motor cortex of BEB patients. Dynamic and symptom-specific structural changes that involve
the primary motor cortex might be an important fingerprint of BEB in comparison to HFS but
also healthy controls, as shown previously. Structural alterations in the primary motor cortex
have been demonstrated in patients with writer’s cramp dystonia by our group [29], and in
spasmodic dysphonia [30].
Fig 1. Cortical thickness differences between the BEB and HFS groups. (A) cross-sectional analysis between BEB
and HFS at baseline; (B) cross-sectional analysis between BEB and HFS after the treatment; (C) longitudinal rate of
change of cortical thickness over time in BEB compared to HFS. Only clusters that survived the P<0.001 threshold are
included. Images are presented at P= 0.05 to better show the extent of cortical changes.
doi:10.1371/journal.pone.0168652.g001
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 6 / 9
Our current analysis underlines further the primary role of pre-SMA in the BEB pathophys-
iology in comparison to HFS subjects. In this region, the rate of cortical thinning was also
much higher and to a greater extent after BoNT than in the HFS group. Pre-SMA is directly
involved in the control of involuntary actions [31] and shows over-activity during dyskinesia’s
in PD patients [32]. Direct electrical stimulation of pre-SMA can elicit an ‘urge’ to move a spe-
cific body part [33]. Neurons in the pre-SMA have been shown to respond during change-of-
plan, altering movement plans [34] and during learning activity for a complex sequence of
hand movements [35]. Hence, structural pre-SMA changes in BEB might be associated with
an altered process of involuntary movement initiation and adaptation to a new order of move-
ments [36]. Thus, its direct structural involvement in the studied patient group is in line with
the functional role of this region. The comparison to the healthy controls did not reveal any
significant changes in the primary motor cortex or pre-SMA indicating these regions is specific
to the structural changes between BEB and HFS patients. First limitation of the study on the
interpretation of the results between the patients and the HC is there is no test-retest reliability
in this study. Second limitation of the study is a higher variance and structural changes at the
time point of maximal symptoms manifestation may preclude detection of treatment effects in
each of the corresponding groups.
After BoNT treatment and with symptomatic improvement, BEB patients showed cortical
thinning in the left inferior temporal, left pre-SMA, right frontal-rostral and primary motor
area. No clear changes of cortical thickness were found in the HFS group after BoNT treat-
ment. The clinical effects of treatment are similar in both groups, namely a decreased rate of
spontaneous muscular contractions in facial muscles, but nevertheless, different structural
changes were found to occur in the analyzed groups. We hypothesize that the achieved immo-
bilization and improvement of the hyperkinetic movement disorder has different effects on
cortical plasticity in the two groups: in BEB a normalization of restructured information flow
through cortico-cortical and basal-ganglia-cortical circuits might occur through immobiliza-
tion which in turn might modify cortical integrity; in HFS BoNT therapy manifests could
merely as a symptomatic effect.
Conclusion
This study shows that standard BoNT therapy brings both clinical improvements as well as sig-
nificant cortical and subcortical reorganization patterns. We hypothesize that BoNT-associ-
ated immobilization of the affected muscles might modify the input in the cortical circuit and
cortico-basal-ganglia loops, which could result in a cortical structural reorganization in order
to adapt to new motoric stimuli.
Acknowledgments
This work was supported by the German Research Council (CRC 1193, project B05). The
funding does not have any involvement in the study design, in the collection, analysis and
interpretation of data, in the writing of the manuscript, and in the decision to submit the arti-
cle for publication.
Author Contributions
Conceptualization: MM SG.
Data curation: HA VCC NK BP.
Funding acquisition: MM SG.
Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 7 / 9
Methodology: HA MM SG VCC.
Resources: GD SG.
Supervision: GD SG.
Writing – original draft: HA MM VCC NK.
Writing – review & editing: BP GD KZ SG.
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Cortical Thickness in BEB
PLOS ONE | DOI:10.1371/journal.pone.0168652 December 16, 2016 9 / 9
