Effect of teriflunomide on cortex-basal ganglia-thalamus (CxBGTh) circuit glutamatergic dysregulation in the Theiler's Murine Encephalomyelitis Virus mouse model of multiple sclerosis
Effect of teriflunomide on cortex-basal ganglia-thalamus (CxBGTh) circuit glutamatergic dysregulation in the Theiler's Murine Encephalomyelitis Virus mouse model of multiple sclerosis
Claire M. Modica 0 1
Ferdinand Schweser 0
Michelle L. Sudyn 0 1
Nicola Bertolino 0
Marilena Preda 0
Paul Polak 0
Danielle M. Siebert 0
Jacqueline C. Krawiecki 0
Michele Sveinsson 0
Jesper Hagemeier 0
Michael G. Dwyer 0
Suyog Pol 0
Robert Zivadinov 0
0 Editor: Ralf A. Linker, Friedrich-Alexander University Erlangen , GERMANY
1 Neuroscience Program, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, United States of America, 2 Department of Neurology, Buffalo Neuroimaging Analysis Center, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, United States of America, 3 Translational Imaging Center, Clinical and Translational Science Institute, University at Buffalo, Buffalo, New York, United States of America , 4 Exercise Science , School of Public Health and Health Professions, University at Buffalo, Buffalo, New York, United States of America, 5 Department of Geology, University at Buffalo , Buffalo, New York , United States of America
changes of the CxBGTh loop in the Theiler's Murine Encephalomyelitis Virus, (TMEV)
Funding: This study was supported by Genzyme,
which is a wholly owned subsidiary of Sanofi.
Research reported in this publication was also
supported by the National Center for Advancing
Translational Sciences of the National Institutes of
Health under award Number UL1TR001412. The
mouse model of MS.
Forty-eight (48) mice were infected with TMEV, treated with teriflunomide (24) or control
vehicle (24) and followed for 39 weeks. Mice were examined with MRS and volumetric MRI
scans (0, 8, 26, and 39 weeks) in the cortex, basal ganglia and thalamus, using a 9.4T
scanner, and with behavioral tests (0, 4, 8, 12, 17, 26, and 39 weeks). Within conditions, MRI
measures were compared between two time points by paired samples t-test and across
multiple time points by repeated measures ANOVA (rmANOVA), and between conditions by
content is solely the responsibility of the authors
and does not necessarily represent the official
views of the NIH. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: Claire M Modica, Ferdinand
Schweser, Michelle L Sudyn, Nicola Bertolino,
Marilena Preda, Paul Polak, Danielle M Siebert,
Jacqueline C Krawiecki, Michele Sveinsson, Jesper
Hagemeier have nothing to disclose. Although we
received funding for the study from a commercial
source: Genzyme, which is a wholly owned
subsidiary of Sanofi, this does not alter our
adherence to PLOS ONE policies on sharing data
and materials (as detailed online in our guide for
competing-interests). Michael G. Dwyer received
personal compensation from Claret Medical.
Robert Zivadinov received personal compensation
from EMD Serono, Genzyme-Sanofi and Novartis
for speaking and consultant fees. He received
financial support for research activities from Teva
Pharmaceuticals, Genzyme-Sanofi, Novartis, Claret
Medical, Intekrin and IMS Health. Dr. Zivadinov
serves on editorial board of J Alzh Dis, BMC Med,
BMC Neurol, Vein and Lymphatics and Clinical CNS
Drugs. He is Executive Director and Treasurer of
International Society for Neurovascular Disease.
independent samples t-test and rmANOVA, respectively. Data were considered as
significant at the p<0.01 level and as a trend at p<0.05 level.
In the thalamus, the teriflunomide arm exhibited trends toward decreased glutamate levels
at 8 and 26 weeks compared to the control arm (p = 0.039 and p = 0.026), while the control
arm exhibited a trend toward increased glutamate between 0 to 8 weeks (p = 0.045). In the
basal ganglia, the teriflunomide arm exhibited a trend toward decreased glutamate earlier
than the control arm, from 0 to 8 weeks (p = 0.011), resulting in decreased glutamate
compared to the control arm at 8 weeks (p = 0.016).
Teriflunomide may reduce possible excitotoxicity in the thalamus and basal ganglia by lowering glutamate levels.
Multiple Sclerosis (MS) is a disease characterized by neurological disability [
] and cognitive
] traditionally characterized by areas of demyelination and inflammation and
brain atrophy. [
] In particular, gray matter (GM) atrophy is associated with cognitive
impairment,  and has a stronger relationship with disease progression and disability than
white matter (WM) atrophy. [
] The understanding of the role of the thalamus in MS has also
gained increasing interest, [
] as its volume reduction correlates with cognitive impairment [
] and fatigue. 
These observations lead to the question of whether disparate GM regions connected by
WM tracts could be pathologically affecting one another. Meningeal inflammation is present
in all stages of MS, [
] and 34% [
] to 50% [
] of cortical MS lesions extend to the pial
surface. Considering that apoptotic neurons are significantly increased in demyelinated cortex in
] the meninges may be involved in the pathological process which affects GM,
suggesting the cortex may be one of the first GM structures involved in the disease initiation.
The cortex-basal ganglia-thalamus (CxBGTh) loop (Fig 1) is crucially influential for motor,
cognitive, and affective behaviors. [13±16] For example, pathology in the substantia nigra of
the basal ganglia causes reverberating problems in the CxBGTh in Parkinson's disease. [
Within this circuit, glutamate, an excitatory neurotransmitter, is highly regulated and linked
to MS disease process in a variety of ways. MS patients with single nucleotide polymorphisms
of the glutamate NMDA receptor 2A subunit domain show more advanced brain atrophy.
] Glutamate levels, as measured by 1H magnetic resonance spectroscopy (MRS), are
increased in acute demyelinated WM lesions. [
] Glutamate transport and metabolism are
decreased in oligodendrocytes around WM lesions in MS.  Excitatory amino acid
transporters, which typically protect neurons and oligodendrocytes from glutamate toxicity, are
downregulated from the membranes of astrocytes in the demyelinated cortex containing
activated microglia. [
] Therefore, dysregulated glutamate within the CxBGTh loop could lead to
parallel and/or exacerbating pathology within structures in this circuit in MS patients.
We used 1H MRS to examine changes in the neurotransmitters glutamate and gamma-ami
nobutyric acid (GABA), and MRI brain volumetric outcomes, in the CxBGTh loop of Theiler's
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Fig 1. Cortex-basal ganglia-thalamus-cortex circuit in mice. Sagittal sketch of representative structures involved in the circuit. A) Cortex
neurons transmit glutamate to the striatum and subthalamic nucleus. Striatum interneurons transmit acetylcholine. B) The striatum transmits
GABA to the globus pallidus externa, globus pallidus interna, and substantia nigra reticulata. The globus pallidus externa transmits GABA to
the subthalamic nucleus, globus pallidus interna, and substantia nigra reticulata. The subthalamic nucleus transmits glutamate to the globus
pallidus interna and substantia nigra reticulata. The substantia nigra pars compacta transmits dopamine to the striatum where some
terminals synapse on upregulating receptors and some on downregulating receptors. C) The globus pallidus interna and substantia nigra
reticulata transmit GABA to the thalamus. D) The thalamus transmits glutamate to the cortex. While these structures and chemicals do not
exist in isolation, dysregulation of any of these molecules may have reverberating effects on the entire circuit. [Figure created with
information from Conn et al. (2005) [
], Arnsten and Rubia (2012) , and the Allen Mouse Brain Atlas [
Murine Encephalomyelitis Virus (TMEV) mice, at pre-disease baseline and after 8, 26, and 39
weeks post-induction (PI). We hypothesized that chronic meningeal inflammation, due to
TMEV infection, would lead to overexcited glutamate neurons in the cortex which could
increase glutamate output to the basal ganglia and thalamus, leading to increased glutamate
and decreased GABA, resulting in increased inhibition of the thalamus and subsequent loss of
volume in the CxBGTh loop structures.
We used TMEV infection because it produces a chronic, immune-mediated condition
similar to MS. [
] The TMEV murine model is characterized by demyelination (largely in the
spinal cord), neuronal death, and reduced remyelination. [
] Infected mice exhibit progressively
decreasing motor coordination [
] which is associated with a development of atrophy in the
brain and spinal cord. [
] Iron accumulation in the thalamus, as measured by the degree
of T2 hypointensity, is also associated with motor impairment.  Meningeal inflammation
occurs throughout the chronic disease course, [
] accompanied by clusters of active
IgG-producing plasma cells. [
] Therefore, the TMEV model of MS could be ideal to investigate the
temporal behavior of the pathological processes in the CxBGTh loop.
Because it was shown that higher glutamate [
] and lower GABA [
] concentrations are
associated with greater physical and cognitive disability and development of brain atrophy in
MS patients, we chose to investigate the effect of an immunomodulatory therapy
(teriflunomide, Aubagio1) that demonstrated a consistent effect on these outcomes in multiple clinical
trials [1, 31±34] on changes of the CxBGTh loop in the TMEV mouse model of MS. Although
teriflunomide is known to selectively and reversibly inhibit dihydro-orotate dehydrogenase,
a key mitochondrial enzyme in the de novo pyrimidine synthesis pathway, leading to a
reduction in proliferation of activated T and B lymphocytes without causing cell death, its
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mechanism of action is not yet fully understood. [
] In addition, previous animal experiment
studies showed that teriflunomide reduced inflammation, demyelination, and axonal loss. [
Against this background, a control-controlled, blinded trial was designed to examine
whether teriflunomide can alter temporal behavior of these pathological processes over 39
weeks. We suspected that teriflunomide could partially reverse glutamatergic dysregulation of
the CxBGTh loop in the TMEV mouse model of MS because it was shown that this treatment
can reduce demyelination and slowdown axonal loss. [35±37]
Materials and methods
Forty-eight (48) 4 to 5 week-old female SJL mice were ordered from Envigo (formerly Harlan
Laboratories, Indianapolis, IN), allowed to acclimate to their new environment for one week,
then given baseline behavioral testing. BHK-21 cell-purified TMEV BeAn 8386 strain working
stock 20A (generously gifted from Dr. Howard Lipton, University Illinois Chicago) [
cultured and titrated by the Genomics/shRNA Shared Resource at Roswell Park Cancer
Institute (Buffalo, NY) following published protocols. [
] 3 106 PFU of TMEV was injected in
0.03mL into the right cerebrum at 7 weeks of age. Following the intraperitoneal (IP) injection
of 50-100mg/kg ketamine and 2-5mg/kg xylazine anesthetic, then intracerebral injection of
TMEV or saline, animals received an IP injection of 2.1mg/kg Yohimbine to counter the
effects of the anesthetic. Mice were then transferred to recover under a warming lamp, and
subcutaneously injected with 1-3ml of saline for recovery. Mice were monitored by technicians
and laboratory staff for 1±3 days post operation to ensure stabilization. Three months after
injection, at the time of peak anti-TMEV antibody levels, [
] blood was collected from mice
via temporal facial vein and tested for presence of anti-TMEV antibodies; all samples were
positive for infection as determined by the Mouse TMEV ELISA Kit (XpressBio, Thurmont, MD).
An additional 10 sham mice injected with 0.03mL of 0.9% saline at 7 weeks of age, which were
not part of the original study design but underwent behavioral testing at the same time points
over a period of 39 weeks, were added to the study for post-hoc comparison purposes.
Following baseline assessments, behavioral testing was conducted on mice at 4, 8, 12, 17,
26, and 39 weeks PI and MRS/MRI examinations at 8, 26, and 39 weeks PI (S1 Fig). Time
points were defined as a period of time up to 7 days before the indicated number of weeks and
up to 7 days after. All behavioral, MRI and laboratory tests and analyses were conducted in a
blinded manner with respect to the treatment status.
All animals were cared for and tested under The Institutional Animal Care and Use
Committee (IACUC) approved protocol (NEU05124Y) by the University of Buffalo. In particular,
mice were clinically monitored daily for signs of gait abnormalities, righting ability, scalding,
and weight. Mice were humanely sacrificed, according to IACUC standards, by cardiac
perfusion following an intraperitoneal injection of 75mg/kg sodium pentobarbital.
Daily administration of treatment by oral gavage began one month PI, at the time when
clinical, histological, and MRI changes, and a dual surge in motor disability, suggest that acute
infection shifts to chronic infection. [
22, 23, 41
] Teriflunomide was given to 24 mice at a
dosage of 20mg/kg in 0.6% carboxymethylcellulose/0.5%, which would be equivalent to a human
] An equivalent volume of identical vehicle solution without teriflunomide was given
to 23 mice (one mouse died prior to initiation of administration). The additional sham cohort
of 10 saline-injected mice received no oral treatment administration.
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Clinical monitoring followed using a scale previously described. [
] Mice were evaluated for
motor disability by rotarod assay (Rotamex-5, Columbus Instruments, Columbus, OH). Mice
were trained on the rotarod for three days immediately preceding the baseline test. Training
on the rotarod consisted of a constant speed of 3 rotations per minute (rpm) for 60s, 4rpm for
60s, and 5rpm for 60s on the first and second days, then a faux trial of the accelerating task (1
to 70rpm at an acceleration rate of 10rpm/m) on the third day. The test trial consisted of an
accelerating task (1 to 70rpm at an acceleration rate of 10rpm/m). At each time point, two
rotarod tests were administered, and the better of the two scores was recorded.
Mice were evaluated for cognitive decline by T-maze continuous alternation task (TCAT),
consisting of free, untimed runs (periods of exploration), before returning to the start position,
in a T-maze. [
] The TCAT allows the mouse to explore one arm of a T-maze per run,
facilitated by blocking entry into the unexplored arm. Upon return to the start arm, the block is
lifted, allowing the mouse to make a new decision of which arm to sample in the following
run. Cognitive performance was quantified by the percentage of alternations, i.e. choosing to
explore the arm which was not chosen in the previous run. One TCAT trial, consisting of 15
runs, was conducted at each follow-up time point, and the percentages of alternations were
recorded. At baseline testing, mice were administered two TCAT trials at least 24 hours apart,
and the better of the two scores was recorded.
MRI acquisition and analysis
All MRI scanning was conducted on a 200mm horizontal-bore 9.4T magnet (Bruker Biospin,
Biospec 94/20 USR) operated with ParaVision (version 5.1; Bruker Biospin) and equipped
with a 440 mT/m imaging gradient system and a cryogenically cooled dual-element transceiver
coil (CryoProbe, Bruker Biospin) placed over the head of the mouse. Induction and
maintenance of anesthesia during imaging was achieved by inhalation of 4±5% and 1±3% Isoflurane.
The total MRI scan protocol lasted approximately three hours. To prevent hypothermia, the
core body temperature was continuously monitored with a rectal probe and regulated with an
embedded water-heating system. Also, the probe head was heated to 37ÊC. Respiration rate
and waveform were continuously monitored and Isoflurane concentration was adjusted if
needed. In order to prevent dehydration, mice were injected with 1mL saline subcutaneously
on an hourly basis. Lubricant was placed on the eyes in order to prevent drying. Respirations
were monitored, and isoflurane was adjusted accordingly to maintain 20±50 breaths per
minute. Following isoflurane administration for MRI scanning, mice were allowed to recover in an
induction chamber under a warming lamp where they received a subcutaneous injection of
2ml of saline. Mice were then transferred under a warming lamp and subcutaneously injected
with saline for recovery. In advanced infection, when clinical signs were exhibited, mice were
given a small Petri plate containing recovery diet gel in addition to normal chow and
subsequently monitored by technicians and laboratory staff for 1±3 days post operation to ensure
MR spectroscopy. The measurement of glutamate and GABA is technically challenging,
because spectroscopic peaks overlap with other metabolites, the metabolite concentrations are
relatively low, they have complex multiplet resonances, and J-coupling evolutions, which impair
accurate quantification with conventional MRS techniques. The special cryogenically cooled
coil used in the present work enabled the direct quantification of glutamate and GABA. [
However, while the widely employed point resolved spectroscopy (PRESS) sequence may in
principle be used for this purpose, its poor spatial localization (>1mm) does not permit
accurate quantification in subregions of the mouse brain as small as, e.g., the thalamus. Here, we
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applied an optimized ultra-short echo time stimulated echo acquisition mode (UTE-STEAM)
sequence with excellent spatial localization (spatial shift less than 260μm) in the cortex, basal
ganglia, and thalamus (2.6ms echo time, 2000ms repetition time, 512 averages, 6000Hz spectral
width, 17min acquisition time; 1.1x1.2x2.9mm3 cortex voxel size, 1.5x1.2x2mm3 basal ganglia
voxel size, 2.1x1.1x1.6mm3 thalamus voxel size; VAPOR water suppression with 250 Hz
] The STEAM sequence provides a substantially improved spatial localization of the
spectroscopic voxel compared to PRESS due to the use of 90Ê instead of 180Ê selection pulses. A
transmit power gain calibration followed by a linear iterative shimming process localized in the
voxel area was performed before each MRS acquisition. The combination of a high magnetic
field strength (high spectral resolution), cryogenic coil (high signal-to-noise ratio) and short-TE
STEAM sequence enabled the direct quantification of GABA and glutamate in small anatomical
sub-regions of the mouse brain.
MRS was acquired in three brain regions of the hemisphere contralateral to the injection
site: cortex, basal ganglia, and thalamus. MRS voxels were prescribed on dedicated high
resolution localizer scans acquired immediately before the MRS (TurboRARE-T2; 3 min
acquisition time). The cortex MRS voxel was placed in the prefrontal area (Fig 2A), with care
used to avoid the skull and the corpus callosum. The basal ganglia voxel was placed in a volume
encompassing the nucleus accumbens, caudoputamen, and globus pallidus, centered primarily
in the caudoputamen (Fig 2B). The thalamus voxel was placed in the most rostral portion of
the thalamus (Fig 2C), with care used to avoid WM tracts.
All spectra were analyzed using LCModel (version 6.3) [
] using an appropriate basis
set for the chosen sequence, phase and eddy current correction. Metabolites with a
CrameÂrRao lower bound value <20% were converted into concentration ratios relative to creatine, a
common internal standard in MRS . To decrease the number of comparisons we examined
the behavior of only glutamate and GABA over time, consistent with our original hypothesis.
Brain volumetry. A multi-echo gradient echo (MEGRE) sequence was used for
volumetric analysis (2.38ms first echo, 4.04ms echo spacing, 9 echoes, 90ms repetition time, one
average, 18Ê flip angle, 27x14.3x8mm field of view, 252x180x100 matrix, 80x80x80μm resolution,
27 minutes acquisition time). MEGRE was preceded by a transmit power calibration
performed in a slice (2 mm thickness) positioned at the top of the brain and a field-map based
2nd-order shimming to the largest possible rectangular portion of the brain while avoiding
regions outside the brain. This shimming process was followed by an iterative linear
shimming. For both shim configurations we assessed the FWHM of the water spectrum and used
the one with the lower FWHM for the final scan. We maximized the anatomical contrast by
Fig 2. MRS voxel placement. A: cortex; B: basal ganglia; C: thalamus.
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Fig 3. Structure segmentation. (A) Image used for segmentation. (B) Manual structure segmentation.
Green: cortex; purple: basal ganglia; blue: thalamus; pink: lateral ventricles.
averaging the magnitude images of all echoes (referred to as ªanatomicalº images in the
following; Fig 3A).
For volumetric assessments, a multi-atlas approach was used. Five individual scans were
selected, covering a range of atrophy levels and brain growth stages. Manual atlases were
created for each of these scans by three independent operators. Regions of interest (ROIs) were
outlined manually using 3D Slicer (www.slicer.org), [
] with the visual aid of Mouse BIRN
Atlasing Toolkit-ready Labeled Atlas v0.6.2. [
] ROIs included the cortex, basal ganglia,
thalamus, and lateral ventricles as a proxy for whole brain (Fig 3B). The cortex encompassed the
entire bilateral cortical layer. The basal ganglia encompassed the bilateral caudoputamen,
bilateral nucleus accumbens, and bilateral globus pallidus. The thalamus encompassed the bilateral
thalamus. The lateral ventricles encompassed the bilateral most dorsal portions of the lateral
To parcellate individual mouse images, the templates were nonlinearly aligned to the target
image with ANTs (Advanced Normalization Tools) symmetric diffeomorphic image
registration with cross correlation. [
] The same deformation fields were then applied to the labeled
atlases corresponding to the templates to bring them into the target space. The appropriate
structure label at each voxel was then selected via a joint fusion weighted voting technique
between all the aligned atlases, taking into account the local voxel-wise correlations with the
corresponding templates.  Better matching templates in each region were therefore more
likely to have their atlas labels selected as the final choice.
Tissue isolation and solochrome cyanine staining
In order to preliminarily assess the effect of TMEV infection on demyelination, coronal brain
sections were stained for myelin with solochrome cyanine stain. [
] Animals (n = 5) from
each treatment arm were intracardiacally perfused with saline, followed by 4% PFA after the
last timepoint scan at 39 weeks. The extracted mouse brain was treated with 6 and 15% sucrose
gradient, flash frozen in dry ice bath and cryosectioned into 16μm thick coronal sections.
Briefly, for solochrome cyanine staining, [
] the sections were placed in Eriochrome cyanine
solution (0.4% ferric chloride, 0.5% H2SO4 and 0.2% Eriochrome Cyanine RS (Sigma) in
distilled water) for 15 mins. Following this, the stain was differentiated using 5.6% ferric chloride
solution (in distilled water) in 30±60 second intervals up to four times, each followed by a
washing step with tap water, until the WM regions were distinctly labelled blue. The images
were acquired on a Zeiss Observer.Z1 microscope using 10x objective and a monochromatic
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camera. The images were pseudo-colored using ImageJ software [
] to restore the original
stain color and emphasize the lesion sites.
Within each treatment arm, clinical scores were compared between two time points by
Wilcoxon paired samples test and across multiple time points by Friedman test; between treatment
arms, clinical scores were compared at each time point by Mann Whitney U test and over
multiple time points by split-plot rmANOVA. Within each treatment arm, rotarod, TCAT,
volume, glutamate and GABA values were compared between two time points by paired samples
t-test and across multiple time points by rmANOVA, and between treatment arms at each
time point by independent samples t-test and over multiple time points by split-plot
rmANOVA. Relationships between change in MRI or MRS and behavioral outcomes were analyzed
by Pearson correlation. Variables determining absolute value change between 0 weeks and 39
weeks were calculated for brain volume, glutamate, and GABA. Pearson correlation
coefficients between these metrics and the final clinical score, rotarod time, or TCAT percentage at
39 weeks were determined.
Data were considered as significant at the p<0.01 level and as a trend at p<0.05 level using
two-tailed tests to minimize spurious findings.
Clinical, motor, and cognitive test findings
One control mouse was removed from the analysis entirely, due to missing most MRI and
clinical time points, bringing the number of control mice down to 23. One additional control
mouse died after baseline during induction procedure; one teriflunomide mouse and one
control mouse died between the 8 and 26 week time points; one teriflunomide and one control
mouse died between the 26 and 39 week time points.
Table 1 provides comparisons of clinical, rotarod, and TCAT scores across treatment arms
for each time point (clinical scores: Mann Whitney U test; rotarod, TCAT: independent
samples t-test). Split-plot rmANOVA found no differences between treatment arms for change in
clinical, rotarod, and TCAT scores over all time points or between any two time points.
Both the control and teriflunomide arms, compared to sham arm, showed higher clinical
scores already at 4 weeks PI, which became significant at 8 weeks, and the differences remained
significant at all time points throughout 39 weeks. Clinical score increased for teriflunomide
and control mice across time (p<0.001, Friedman test). From 4 weeks to 39 weeks, clinical
scores increased between each time point within each condition (p<0.01, Wilcoxon), with the
exception of 17 to 26 weeks in teriflunomide mice, which did not exhibit a significant
deterioration (p = 0.097, Wilcoxon). We observed that both teriflunomide and control cohorts were
rendered spastically paralyzed with an obvious loss in posture by the end of 39 weeks. Both
teriflunomide and control mice presented a waddling gait with a lowered rear at 4±8 weeks, and
severe waddling gait and eventual hind limb spastic paralysis progressed over the 39 weeks of
TMEV infection. Further, the sham arm did not exhibit any severe clinical disability during
the study, though a slight lowering of the tail and rear end was observed in some cases at 26
and 39 weeks
Rotarod scores did not change for either teriflunomide or control condition as indicated
over time by rmANOVA or between time points by paired samples t-test. However, there
were several trends in both arms indicating a drop in score between 0 and 4 weeks (control
p = 0.055; teriflunomide p = 0.038, paired samples t-test) and an increase in score between 4
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Clinical is given in scores, rotarod in seconds, and TCAT in percentage. The TCAT T shape provides the mice with an untimed opportunity to explore left or
right arms. Upon selection of an arm the unexplored arm was blocked off to return mouse to start. 15 trials were performed per session and left/right (L/R)
arm selection was noted. Scores were determined at a percent ratio over 15 trials, with 1 for alternation and 0 for repetition. The statistical analysis was
performed using Mann Whitney U test for clinical sores and independent samples t-test for rotarod and TCAT values. One control mouse was removed from
the analysis entirely, due to missing most MRI and clinical time points, bringing the number of control mice down to 23. One additional control mouse died
after baseline during induction procedure; one teriflunomide mice and one control mouse died between the 8 and 26 weeks times points; one teriflunomide
and one control mice died between the 26 and 39 weeks' time points.
and 8 weeks (control p = 0.023; teriflunomide p = 0.054). At baseline and at 4 weeks, both
teriflunomide and control arms showed lower rotarod score, compared to the sham arm.
TCAT scores decreased in both conditions over time (p<0.01, rmANOVA), while post-hoc
paired samples t-tests did not detect any changes between any two time points in either
condition. However, there was a trend toward decreasing TCAT values between 0 and 4 weeks in
both control (p = 0.019) and teriflunomide (p = 0.038, paired samples t-test) arms. There was
also a trend toward decreasing TCAT values between 17±26 weeks in control (p = 0.035), but
not in the teriflunomide arm (p = 0.225, paired samples t-test). Over 39 weeks, no TCAT
differences were observed between teriflunomide and control arms compared to the sham one,
except a decreased value in the sham arm at 12 weeks.
Fig 4 shows glutamate ratios relative to creatine and phosphocreatine concentration and
independent samples t-test p-values across treatment arms at each time point. No significant
differences were detected with regard to glutamate concentrations over all time points using
rmANOVA or between any two time points between treatment arms, and independent sample
t-tests found no differences between treatment arms at any single time point.
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Fig 4. Glutamate and GABA change over time across conditions. A-C: glutamate; D-F: GABA. Brackets indicate independent samples
t-test at a single time point; arrows in bars indicate direction of change from previous time point in paired samples t-test: *p<0.01, ²p<0.05;
orange = control, blue = teriflunomide.
Cortex. Glutamate in the cortex decreased over time within each treatment arm (p<0.001,
rmANOVA) (Fig 4A). In control mice, this was largely driven by a decrease between 26 to 39
weeks (p<0.001, paired samples t-test), while in teriflunomide mice, the decrease was gradual
across time with no significant changes between any time points as indicated by paired samples
Basal ganglia. There were no changes over time in either treatment arm with regards to
glutamate in the basal ganglia, though the control arm trended toward decrease between 26 to
39 weeks (p = 0.033) while the teriflunomide arm trended toward decrease between 0 to 8
weeks (p = 0.011, paired samples t-tests), which resulted in a trend toward difference between
the two treatment arms at 8 weeks (p = 0.016, independent samples t-test, Fig 4B).
Thalamus. While there were no changes over time in either condition with regards to
glutamate concentration in the thalamus, there was a trend toward change in the control arm
(p = 0.034, rmANOVA), largely driven by a trend toward increase between 0 to 8 weeks
(p = 0.045, paired samples t-tests), resulting in a trend toward increased glutamate between
two treatment arms at 8 and 26 weeks (p = 0.039 and p = 0.026, independent samples t-tests,
Fig 4 shows GABA ratios relative to creatine and phosphocreatine concentration and
independent samples t-test p-values across treatment arms at each time point. No significant differences
10 / 19
were detected with regard to GABA concentrations over all time points using rmANOVA or
between any two time points between treatment arms, and independent sample t-tests found
no differences between treatment arms at any single time point.
Cortex and thalamus. GABA concentrations were stable over time by rmANOVA or
between time points by paired samples t-test in either treatment arm, both in the cortex and
thalamus (Fig 4D and 4F). In the basal ganglia, GABA changed over time in the teriflunomide
arm (p<0.001, rmANOVA), driven largely by a decrease between 8 to 26 weeks (p<0.001,
paired samples t-test), followed by a significant increase between 26 to 39 weeks (p = 0.002,
paired samples t-test, Fig 4E).
Basal ganglia. GABA in the basal ganglia only showed a trend toward change over time in
the control arm (p = 0.015, rmANOVA), but this was reflected by a decrease between 8 to 26
weeks (p = 0.002, paired samples t-test) and additional trend toward decease between 0 to 8
weeks (p = 0.032, paired samples t-test). While the decrease in basal ganglia GABA from 8 to
26 weeks trended toward being larger in the teriflunomide than in the control arm (p = 0.014,
split-plot rmANOVA), there was notably no subsequent increase in GABA in the control arm
as indicated by paired samples t-test.
Brain volume findings
Fig 5 shows brain volume assessments of the examined structures in mm3 and independent
samples t-test p-values across conditions at each time point. With regard to changes in brain
volume measures, no significant differences between treatment arms over all time points, or
between any two time points, were found by rmANOVA, and independent sample t-tests
found no differences between conditions at any single time point.
Cortex. Volume decreased in the cortex over time in the teriflunomide condition
(p<0.001, rmANOVA) (Fig 5A), and trended toward decreasing in the control condition
(p = 0.013, rmANOVA). In the teriflunomide arm, this decrease was evident between 0 to 8
weeks (p = 0.006) and between 26 to 39 weeks (p<0.001), and trended toward decrease
between 8 and 26 weeks (p = 0.036, paired samples t-test). In the control arm, there was a
decrease only between 26 and 39 weeks (p = 0.008, paired samples t-test).
Basal ganglia. Brain volume did not change in either treatment arm in the basal ganglia,
but the control arm trended toward increase across time and between 0 to 8 weeks (p = 0.01,
rmANOVA; p = 0.025, paired samples t-test; Fig 5B).
Thalamus. Volume increased in the thalamus within each treatment arm (p<0.001,
rmANOVA). In the control arm, this increase was driven between both 0 to 8 weeks (p<0.001) and
8 to 26 weeks (p = 0.001, paired samples t-tests, Fig 5C). These increases were not observed in
the teriflunomide arm, resulting in a trend toward difference between conditions in change
from 0 to 8 weeks (p = 0.02, split-plot rmANOVA), although there was a trend toward
increasing between 8 to 26 weeks (p = 0.015, paired samples t-test).
Lateral ventricles. Volume increased in the lateral ventricles within each condition
(p<0.001, rmANOVA), largely driven by an increase between 0 to 8 weeks within each
condition (p<0.001, paired samples t-test, Fig 5D).
Associations between behavioral outcomes and changes in MRI and
MRS over 39 weeks
Negative associations were found between change in cortex volume (R = -0.664, p = 0.005, Fig
6A) and thalamus volume (R = -0.645, p = 0.007, Fig 6B) and clinical score in the control arm,
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Fig 5. Volumetric changes over time across conditions. Arrows in bars indicate direction of change from previous time point in paired
samples t-test: *p<0.01, ²p<0.05; orange = control, blue = teriflunomide.
indicating that as those volumes decreased, clinical outcomes worsened. Neither of these
associations were significant in the teriflunomide arm, though one was trending (R = -0.355,
p = 0.148, Fig 6A and R = -0.531, p = 0.023, Fig 6B, respectively). There were also trends
toward a negative association between change in basal ganglia GABA and clinical score
(R = -0.616, p = 0.011, Fig 6C). This was not observed in the teriflunomide arm (R = -0.187,
p = 0.458, Fig 6C).
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Fig 6. Relationships between change in MRI/MRS and clinical and rotarod score outcomes. A-C:
clinical score; *p<0.01, ²p<0.05; orange = control, blue = teriflunomide.
Preliminary histological findings
We detected that both control and teriflunomide treatment arms exhibited demyelinated
lesions in the corpus callosum (Fig 7). We did not observe demyelination in the basal ganglia.
In a study of 48 mice infected by TMEV, we found that intervention with teriflunomide
treatment did not significantly change the course of behavioral outcomes or MRI volumetric
changes across the 39 weeks. Teriflunomide partially modulated glutamate changes in the
thalamus and basal ganglia in the early/mid stage of the disease. There were different associations
between clinical and MRI/MRS outcomes in the TMEV model of MS of the two treatment
Fig 7. Solochrome staining TMEV induced demyelination in treated animals. A and B show images of coronal sections from
teriflunomide and control animals, respectively. The arrows point at some of the demyelinated regions within the corpus callosum of each
animals. Note that solochrome stain is not saturated, this is indicative of some level of demyelination even in the stained areas of the corpus
callosum. The scale bar is 0.75mm long.
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Mice under the treatment of teriflunomide exhibited patterns of trends toward earlier
glutamate decrease in the basal ganglia. The control arm exhibited a trend toward glutamate
increase in the thalamus that was not evident in the teriflunomide arm. This suggests that
teriflunomide may be inducing early reduction of glutamate in the basal ganglia, and may be
preventing early glutamate increase in the thalamus, possibly resulting in an overall lower
concentration during the chronic infection. In combination with cross-sectional trends toward
decreased concentrations of glutamate in teriflunomide arm compared to the control arm,
these patterns may suggest that control mice were more likely than teriflunomide to exhibit
higher levels of glutamate in the CxBGTh circuit at varying time points throughout chronic
infection. These data indicate that teriflunomide intervention may help to partially promote
neuroprotection by reducing possible excitotoxicity.
We found that there was increased glutamate and decreased GABA concentrations in the
basal ganglia, likely leading to an environment of increased glutamate concentration in the
thalamus. In line with our hypothesis, we showed that glutamate dysregulation in one of the
structures of the CxBGTh could contribute to pathology in the entire circuit in the TMEV
model of MS. A future histopathological analysis of the animals from the current sample
(preserved) will aim to elucidate these initial MRS findings. In both treatment arms, GABA
decreased between 8 to 26 weeks in the basal ganglia, however teriflunomide induced an
increase in GABA in the chronic infection stage, and limited reduction of GABA in the early
phase of the disease. Being an inhibitory neurotransmitter, elevated GABA may play a role in
preventing excitotoxic conditions, particularly in an environment in which glutamate is
There were no volumetric differences between the two treatment arms over the follow-up.
The only trend between treatment arms was evidenced between 0 to 8 weeks in the thalamus,
with control arm increasing more in volume than the teriflunomide arm. These volumetric
findings may be explained by greater TMEV-induced inflammation in the control arm, and by
decrease of early acute inflammation in the teriflunomide arm, a phenomenon well described
in MS patients, as ªpseudoatrophy effect.º [
] Alternatively, increased inflammation in the
control arm may have masked tissue structure loss until the later stages of chronic infection.
Either scenario would also explain the trend toward increase in the basal ganglia volume
exhibited by the control arm, but not the teriflunomide arm, between 0 and 8 weeks. The increase
in lateral ventricle volume across all mice suggests that the infection leads to either atrophy
and/or a reduction in tissue growth, and that this occurs during acute infection and/or the
early part of chronic infection. Teriflunomide intervention also led to an earlier pattern of
cortical volume decrease, with the teriflunomide arm decreasing, or trending toward decrease,
between every time point but the control arm decreasing only between the final time points. A
preliminary histological analysis confirmed demyelinated lesions in the corpus callosum, but
not in the basal ganglia. More sophisticated histological analysis on the effect of the
teriflunomide on myelination levels, oligodendrocyte density, maturity and microglial density will be
presented in the future.
While both control and teriflunomide treatment arms exhibited significantly higher clinical
progression compared to the sham arm, starting from 4±8 weeks of the follow-up, they showed
similar clinical progression. It is notable that between 17 and 26 weeks, the teriflunomide arm
did not exhibit significant progression while the control arm did. This was also the only pair
of time points within the chronic infection period in which the control arm trended toward
decline in TCAT performance while the teriflunomide arm exhibited no decline. While
teriflunomide did not prevent disease progression, it may have slowed it down, particularly during
this time period. We chose one month PI to begin administration of teriflunomide, however, it
may be more effective to begin administration of an immunomodulatory agent at clinical sign
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onset, around two months PI. In a previous study we conducted, [
] we used RT-PCR to
quantify TMEV viral titer in the brain and spinal cord and, even though our sample was small,
we found at 21 days PI that the spinal cord-to-brain proportion of virus (0±10 fold greater)
was smaller than the 46±124 days PI spinal cord-to-brain proportion (10±100 fold greater)
found by Trottier et al. (2002) using the same method. [
] This suggests that viral load may
migrate from heavy in the brain and light in the spinal cord to light in the brain and heavy in
the spinal cord over time. A robust immune system may be necessary up-front, during acute
infection and into the early part of chronic infection, in order to combat the heavy viral load in
the brain, while an effort to decrease chronic inflammation would have greater beneficial effect
after the viral titer has declined.
The trend toward decline in TCAT performance in the control arm may be the first
evidence suggesting cognitive decline within the chronic infection period in TMEV, although no
significant differences were found respect to the sham arm over 39 weeks. The spatial
component of TCAT involves spontaneous alternation that is driven by an intrinsic motivation to
seek out and explore novel space. [
] TCAT involves coordination between several brain
regions including; the cortex, thalamus, substantia innominate, as well as the cerebellum. [
For the purpose of this study, we sought to measure the general cognitive capabilities and
how TMEV infection and treatment with teriflunomide affected performance, including the
regions suggested previously to be involved in the TCAT task.
Rotarod testing for motor ability did not show decline in the chronic infection period, but
rather a stagnation of performance, and there were no differences respect to the sham arm
performance. Previous studies demonstrated an improvement of performance on similar versions
of rotarod assays in healthy control mice, [
] which was observed also in current study,
and which could suggest that a lack of improvement in TMEV arms in our assay may be due
to the effect of the infection on motor ability. Given that sham arm was not part of the original
study design, and was added for post-hoc comparison purposes in a second time, future work
will need to test a larger group of healthy control mice in order to determine predictable
improvement patterns, or the assay may need to be modified. The assay may need to be more
challenging in order to produce a better resolution between animals of slightly varying ability,
or training for the rotarod assay could be more robust in order to reduce the practice effect
caused by repeated follow-up testing. In addition, inclusion of more frequent MRI time points
and histological analysis could characterize better acute vs. chronic stage of TMEV infection in
the first 8 weeks of follow-up after PI.
These MRS and MRI findings may help to explain why cognitive and clinical scores in the
teriflunomide arm did not decline in the same fashion as the control condition between 17
and 26 weeks. If teriflunomide is able to partially reduce excitotoxicity and inflammation in
the CxBGTh circuit, an easing of clinical progression may just be beginning to show at 17
weeks, or three months after the start of drug administration, which is in line with human
findings, indicating that it takes approximately 3 months for teriflunomide to reach the steady
] This is also supported by the association analyses, which found differences in
associations between the two treatment arms, suggesting that teriflunomide intervention may
be able to alter TMEV-induced pathology in the CxBGTh circuit and subsequent behavioral
While future studies are needed to determine the structural volume and metabolite changes of
healthy control vs. TMEV-infected mice, we can conclude that teriflunomide may partially
affect glutamate regulation in the TMEV model of MS, in a fashion that reduces the likelihood
15 / 19
of early/mid-term excitotoxicity in the CxBGTh circuit. Because glutamate dysregulation is
associated with MS, it is possible that teriflunomide also reduces the propensity to
exictotoxicity in the CxBGTh in MS, and therefore future MRS studies on the CxBGTh circuit in MS
should be conducted using a variety of disease-modifying drugs.
S1 Fig. Experimental timeline. MRI was conducted at 0, 8, 26, and 39 weeks. Rotarod and
TCAT were conducted at 0, 4, 8, 12, 17, 26, and 39 weeks. Therapeutic intervention began at 4
We are grateful for the assistance of Doug Weston, Trina Rudra, and John Barbieri in helping
to take care of the mice involved in this study. We thank Bill Macy, Gary Olsen, and Simon
Peng at the University at Buffalo Engineering Machine Shop, who helped construct the
Tmaze. We also thank Dr. Irwin Gelman and Renae Holtz, who produced the virus at the
Genomics/shRNA Shared Resource at Roswell Park Cancer Institute, which is funded by NCI
P30CA16056 and RPCI Core Grant.
Conceptualization: Robert Zivadinov.
Data curation: Claire M. Modica.
Formal analysis: Paul Polak, Jesper Hagemeier.
Funding acquisition: Robert Zivadinov.
Investigation: Claire M. Modica, Ferdinand Schweser, Michelle L. Sudyn, Nicola Bertolino,
Marilena Preda, Danielle M. Siebert, Jacqueline C. Krawiecki, Michele Sveinsson.
Methodology: Michael G. Dwyer.
Project administration: Claire M. Modica.
Resources: Claire M. Modica.
Software: Paul Polak.
Supervision: Ferdinand Schweser, Robert Zivadinov.
Validation: Robert Zivadinov.
Visualization: Claire M. Modica.
Writing ± original draft: Claire M. Modica.
Writing ± review & editing: Claire M. Modica, Suyog Pol, Robert Zivadinov.
16 / 19
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