Clinical trial NCT03373162
Conditions: Healthy
Sponsor: University of California, Irvine
Start date: 2017-09-30; Completion date: 2018-03-16
Enrollment: 10 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The injection of BOTOX into peripheral muscles has been shown to have therapeutic effects in a growing number of indications including disorders involving skeletal muscle (e.g., strabismus, blepharospasm, cervical dystonia, spasticity, cosmetic), smooth muscle (e.g., bladder), glands (axillary hyperhidrosis) and nociceptive pain (e.g. migraine) (Brin. 2014). Recently, several studies have suggested that peripheral BOTOX injections in the region of the glabellar lines (muscles around the eyes) may be effective in treating major depression and chronic migraine. However, the mechanism underlying the effect of peripheral BOTOX injections on the brain is not well understood. Therefore, the Investigators propose to further explore the functional effects of BOTOX injections on brain in healthy controls. The approach will involve the measurement of brain metabolites in the brainstem using MRS as well as an investigation of the functioning and connectivity between regions of the brain using resting state MRI and high resolution fMRI.

Detailed description:
Participants will receive one MRI scan session prior to a standardized, clinical dose of BOTOX injected into the forehead per FDA-approved procedures. Participants will then undergo a second MRI scan session 2-3 weeks later, after the BOTOX has taken maximal effect. Examining fMRI of the brainstem and the functional connectivity of the brainstem and connections to cortex, may provide additional insight into the effects of BOTOX on neurologic and psychiatric disorders.

Arms: arm 1: Participants will receive MRI scans pre and post-Botox injection, including magnetic resonance spectroscopy, structural, and functional MRI.
Interventions: intervention 1: onabotulinumtoxinA
Intervention descriptions: intervention 1: This study is a pre- and post-design. One scan will be collected prior to BOTOX injection and the second will be collected 14-21 days post-injection. BOTOX injections will be limited to 20 units in the glabellar area, as approved by the FDA .

Protocol documents:
Prot_000.pdf: 
1 
 
Last modified: January 10, 2017 
 
 
 
 
 
 
 
 
Measuring the functional effects of BOTOX on the 
brainstem using MR Spectroscopy (MRS) and fMRI 
 
PI: Craig Stark, Ph.D., Professor 
 
 
 
 
 
 
 
320 Qureshey Research Lab 
Department of Neurobiology & Behavior 
University of California, Irvine 
Irvine, CA 92617 
(949) 824-4230 
cestark@uci.edu 
 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
2 
Abstract 
The injection of BOTOX into peripheral muscles has been shown to have therapeutic effects 
in a growing number of indications including disorders involving skeletal muscle (e.g., 
strabismus, blepharospasm, cervical dystonia, spasticity, cosmetic), smooth muscle (e.g., 
bladder), glands (axillary hyperhidrosis) and nociceptive pain (e.g. migraine) (Brin. 2014). The 
mechanism of action on peripheral muscle is established, and the effects on peripheral 
sensory nociceptors continue under active investigation (Burstein et al., 2014; Zhang et al., 
2016).  Recently, several studies have suggested that peripheral BOTOX injections in the 
region of the glabellar lines (corrugator and proceris muscles) may be effective in treating 
major depression. In addition, peripheral cranio-facial injections have been found effective in 
treating chronic migraine (Whitcup et al, 2014). It is established that peripheral interventions 
can modulate functional CNS connectivity in motor and sensory disorders (Cook et al., 2014; 
DeGiorgio et al., 2013).  In addition, functional imaging has demonstrated that modifying 
eyebrow height or corrugator contraction activity can have functional changes at the level of 
the amygdala (Heller et al., 2011; 2014; Hennenlotter et al., 2009; Kim et al., 2014).  The 
mechanism underlying the effect of peripheral BOTOX injections on afferent systems is not 
well understood.  Nevertheless, brainstem pathways are integral in this setting as the VIIth 
cranial nerve provides efferent innervation to the target muscles and sensation is mediated by 
the Vth cranial nerve.  We propose to further explore the functional effects of BOTOX 
injections on brain in healthy controls. Our approach will involve the measurement of brain 
metabolites (precursors of metabolism) in the brainstem using MRS as well as functional 
connectivity (resting state MRI) and functional activation from sensory motor tasks using high 
resolution fMRI. Examining fMRI of the brainstem and the functional connectivity of the 
brainstem and connections to cortex, may provide additional insight into the effects of 
BOTOX on neurologic and psychiatric disorders. 
Overview of fMRI 
Functional MRI (fMRI) allows the non-invasive visualization and mapping of human brain 
function (Friston et al., 1999, Francis et al., 2000, Jancke et al., 2000, Muller et al., 2009). 
Neural activity in functioning areas of the brain reduces local oxygen concentration, resulting 
in subsequent increase in blood flow (to replenish the cells with oxygen). This oxygenated 
blood creates a blood oxygen level dependence (BOLD) effect on the MR signal.   By this 
BOLD effect, one can indirectly detect where cerebral metabolism is taking place. 
There are few fMRI studies investigating the effects of BOTOX that have demonstrated 
relationships between facial feedback and emotional stimuli in the amygdala (Kim et al. 2014, 
Hennenlotter et al. 2008).  These results suggest that motor input from facial expressions 
plays a role in perception of emotion.  In fact, there are numerous current theories that 
peripheral motor function is intrinsic to all cognitive processing.  By modulating peripheral 
input into the brainstem by relaxing facial muscles innervated by the brainstem nuclei, it is 
possible that reduced tonic activation (from the facial muscles) will affect other neural network 
thresholds as well, promoting functional plasticity, and leading to quantifiable differences in 
the fMRI signal. 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
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Overview of MRS 
Magnetic resonance imaging spectroscopy (MSI/MRS) is used to quantify the level of various 
metabolites (intermediary and byproducts of metabolism) in the brain. Many of these 
metabolites can be measured using conventional proton (1H) imaging or with specialized 
Phosphorous (31P) techniques. MRS yields data from an arbitrary-sized voxel in the form of a 
spectrum, from which metabolic concentrations can be determined (Figure 1). These 
concentrations can then be compared across groups or related to individual differences in 
behavior, treatment response, etc. 
For example, a number of metabolites index neural activity and can be used to dissociate 
excitatory from inhibitory activity. Total Creatine (tCr) signal in MRS is a combination of both 
phosphocreatine (PCr) and creatine (Cr). Both act as ATP regulators and their concentration 
in the brain is reflective of the high-energy phosphate buffer system. Glutamate (Glu) is the 
most abundant metabolite in the brain and a dominant excitatory neurotransmitter, released 
during neuronal excitation and converted to Glutamine (Gln). The Glutamate-Glutamine cycle 
is highly energy demanding and readily observed.  GABA is the most important inhibitory 
neurotransmitter in the brain and, while more difficult to measure with MRS, can be accurately 
quantified with specialized protocols. GABA levels have been shown to change during motor 
learning (Floyer-Lee et al, 2006). In addition, elevated GABA levels have been detected in 
migraine (Aguila et al. 2015), and depression (Licata et al. 2014) making this metabolite well 
suited for this work.  
Other metabolites that can be measured index cellular energetics in the form of phospholipid 
precursors, metabolites, and high energy phosphates.  A 31P MRS scan, for example, can 
measure the phosphocreatine (PCr) and ATP (two doublets and a triplet) resonances with 
their ratio frequently used as a tissue-energy status indicator.  Similarly, the 
phosphomonoester (PME) and phosphodiesters (PDE) peaks represent precursors of 
membrane synthesis and breakdown products. 
Finally, several of our target metabolites are linked to cellular structure and, as such, are often 
found to be markers of pathology. N- Acetyl-Aspartate (NAA) in the brain is localized to the 
neuronal bodies, axons and dendrites and is hence considered a marker for neuronal integrity 
and function.  Studies of disease involving loss of neurons (stroke, epilepsy, tumors, multiple 
sclerosis) have shown a consistent decrease of NAA. Additionally, the choline peak in the 
brain consists of Choline (Cho), Phosphocholine (PCho) and Glycerophosphocholine 
(GlPCho) and reflects cellular density and membrane breakdown.  
Our proposed work uses cutting edge techniques to investigate whether effects of BOTOX 
can be observed in the CNS.  The techniques will maximize the number of detectable 
metabolites by including not only traditional 1H MRS, but eventually also including 31P-based 
MRS. 
Preliminary Data 
Our research group has significant experience in a wide range of MRI techniques, including 
MRS. As we have only recently begun MRS scanning on the 3T Philips magnet at UC Irvine, 
we present here sample data from this scanner to demonstrate its viability.  Demonstrations 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
4 
of functional, structural, and diffusion-based MRI on the UCI scanner are available in our 
published work (e.g., Bennett and Stark, 2015, Stark et al. 2015, Mattfield and Stark 2015, 
Yunshkevich et al. 2015, Huffman and Stark 2014, Lacy et al. 2012).  
Sample 1H MRS data  
To demonstrate our ability for 1H MRS in the 
hippocampus, brain stem and posterior 
cingulate, we have collected pilot data using 
our 3T Philips MRI in combination with a 32-
channel head coil. 1H spectroscopy data 
were acquired using the PRESS localization 
sequence 
(Bottomley, 
1984; 
TR/TE 
= 
2000/37 ms), from a 2.5x1.5x1 cm voxel in the left and right hippocampi of all subjects 
following an established protocol (Valenzuela et al, 2003). For the posterior cingulate, a larger 
voxel measuring 2x2x2 cm was selected. Due to its smaller location, the brainstem voxel 
measured 1.5x1x1 cm All spectra were processed using the TARQUIN software (v.4.3.6) for 
NMR/MRS spectra quantitation (Wilson et al., 2011) and a sample is shown in Figure 1. 
Preliminary 1H MRS results are shown in Figure 2 and are remarkably similar to prior work 
(Bartha et al., 2008).  This measurement is shown in the hippocampus but can be done in any 
other brain structure, including the brainstem and the posterior cingulate. 
Sample SV-PRESS (1H) data for GABA  
In addition to the standard metabolites measured with short 
TE (37ms) PRESS, we now have the capability for improved 
GABA measurements by choosing the combination of 
TE1/TE=15/105 ms results in most optimized parameters to 
resolve the 1.89 and 2.28 ppm peak of the GABA spectrum. 
We are currently testing and tuning its parameters. 
High resolution fMRI of the Brainstem 
Our laboratory routinely collects high resolution fMRI of the medial temporal lobe for memory 
and aging studies. Coincidentally, our protocol is also ideally oriented to observe fMRI of the 
brainstem. Initial results with high resolution (1.5mm) fMRI of the brain stem are shown in 
Figure 3. 
 
 
 
 
Fig 3. (Left) high resolution fMRI image, before spatial smoothing with corresponding time series 
(TR=2). On right, high resolution T2 image of brainstem for region of interest registration. 
 
Fig 1: Characteristic hippocampal spectra for TE 37ms 
(left), TE1/TE 15/105ms (mid), posterior cingulate 
TE=37ms (right), and for brainstem TE=35 ms (far right) 
Fig 2: Our hippocampal SV PRESS 
(N=9, red) and from Bartha et al. 
(2008, blue)  


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
5 
 
Hypotheses 
There are two goals of the proposed preliminary work.  The first is to determine whether there 
are metabolic differences in the brain stem in healthy individuals as a result of peripheral 
administration of BOTOX into the glabellar muscles.  This is an open question that can be 
addressed with MRS.  While the doses used in cosmetic treatments are low, an effect via the 
trigeminal nerve is certainly possible and knowing whether such an effect exists in this cohort 
would be highly relevant for attempts to understand the effects of BOTOX on disorders such 
as migraine or depression.  Importantly, we proposed to diverge from typical MRS here and 
to employ “functional MRS” by collecting MRS data during the performance of an 
emotionally-arousing task.  If BOTOX is altering emotional processing in the brain via a “facial 
feedback” mechanism, altered metabolite levels in the brainstem might be resolved. 
The second goal is to better understand the effect of BOTOX on functional activity (measured 
using fMRI) in the brain and in the brainstem.  Consistent with prior work, we propose that 
BOTOX will alter emotional-related activity in the amygdala (Kim J, 2014).  We propose to 
extend this work in several ways by measuring fMRI activity in the brainstem as well and 
understanding its relationship to activity in structures such as the amygdala.  Again, if BOTOX 
is altering emotional processing via an alteration of the facial feedback mechanism, this could 
be resolved either as an alteration of fMRI activity levels or as an alteration in “functional 
connectivity” (correlation of activity over time) between the brainstem and the amygdala.  
Proposed Protocol: Pilot Study on Healthy Controls 
Our aim is to be able to examine the effects of BOTOX on the CNS to determine potential 
mechanisms for the effects of BOTOX on disorders such as migraine and depression. To 
date, there are no MRS studies of BOTOX to guide the investigation. We therefore propose a 
staged design with an initial pilot study in healthy controls undergoing BOTOX treatment for 
cosmetic purposes, potentially followed by an expanded study in patients being treated for 
migraine or depression. Additionally, in this initial pilot, we will explore the feasibility of 
brainstem fMRI in those two groups of participants. The connectivity between regions in the 
brainstem will be studied in the presence of angry/happy faces paradigm and at rest. Possible 
connections with metabolite changes will be explored as well. 
Participants: We propose to scan 10 healthy, right handed females. Because the sample size 
is so small, we will be constraining the inclusion/exclusion criteria to minimize variability. 
Participants will be naïve BOTOX users (e.g. no previous use of BOTOX), ages 30-40 years 
old. Participants will be excluded for the presence of neurological or psychiatric conditions or 
the presence of any risk factors for MRI. Participants will be recruited from UCI and/or the 
surrounding community via e-mail blasts and word-of-mouth. They will receive $50 
compensation per MRI scan and the cosmetic BOTOX will be provided by Allergan. 
Design: We propose a pre- and post-design. One scan will be collected prior to BOTOX 
injection and the second will be collected 2-4 weeks post-injection. BOTOX injections will be 
limited to 20 units in the glabellar area, as approved by the FDA and administered by a 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
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certified clinician at the UC Irvine Medical Center (ENT Satellite Office in Irvine). Each 
scanning session will last approximately one hour and include: 
1) T1 MPRAGE (3D whole-brain structural image) = 5 min 
2) T2 localizers (axial, coronal and sagittal) = 6 minutes 
3) Resting state fMRI = 7 minutes  
4) fMRI (reduced TR and FOV) =15 min  
5) MRS–SV of brain stem = 7 min  
 
MPRAGE: This is a standard 3D structural image of the whole brain that will allow us to 
localize all fMRI findings as well as to determine whether there are any pre-existing cortical 
abnormalities in any participants.  While we do not expect to observe any pre- vs. post- 
structural changes regional brain shape or size, we will be able to assess these factors. 
Resting-state fMRI: We will collect whole-brain fMRI data for 7 min of eyes-open resting 
state fMRI. This data is commonly used to reveal patterns of network-level connectivity (and 
group-level changes in connectivity) in the brain (Power et al., 2011). Changes in resting 
states between groups (with and without BOTOX) would likely be observed. We will examine 
the frequency of switching of modes (unique waveforms from multi-component analysis) 
between groups. We will also explore the functional connectivity between dura-sensitive 
neurons and their projections to the hypothalamus, thalamus and mid-brain, as shown by 
Burstein (Burstein et al.,1998).  
fMRI: The volunteers will participate in an fMRI scanning session following the protocol of 
Kim et al. 2014. Photos of series of angry, happy and surprised facial expressions (consisting 
of 9 male and 9 female identities) will be presented for 17, 50 and 1000 ms followed by black 
and white pattern, presented for 250 ms (serving as a retinal wipe). During each trial, 
participants will be asked to report using a button box whether they thought each of the faces 
they saw represent a positive or a negative emotion. We will analyze fMRI data of the 
brainstem following a modified method used by Beissner et al. 2011.  
MRS-1H This will serve as an exploratory study to quantify a series of standard metabolites 
(described above) from a voxel located in the brainstem. If BOTOX injections invoke plasticity 
in the afferent motor pathways, the MRS data acquired may detect altered Glutamate or 
GABA concentrations, as GABAergic systems relate to short term motor learning and 
behavior (Stagg et al. 2011, Floyer-Lea et al. 2006). Differences in metabolite concentrations 
in brainstem following the fMRI task may show a link between this region and the amygdala, 
where those activations have been shown to occur (Kim et al. 2014). These concentrations 
may be further modulated in the presence of BOTOX injection. 
Problems and Alternatives:  
Our preliminary data of 1H-MRS of the hippocampus, brainstem and posterior cingulate have 
yielded results comparable with other published studies. However, the small voxel sizes used 
in our preliminary data yield spectra with moderate noise. This can be improved by increasing 
the voxel sizes in line with other literature. Recent advances have provided the means to 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
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measure hard to resolve peaks (such as GABA) through the use of multi echo PRESS 
parameter selection should mitigate the need for new and expensive hardware and software. 
Additionally, fMRI of the brainstem is not common and some claim it poses some additional 
difficulty due to increased physiological noise (Beissner et al. 2011). Our high resolution fMRI 
data of the hippocampus/brainstem do not seem to be suffering from these issues, but it is 
possible that the thicker slices and larger voxel sizes used in whole brain (standard-resolution) 
scans will. We will conduct tests on several existing datasets in our lab and others to 
determine the magnitude of any such effects and will adjust to higher-resolution oblique 
coronal images (covering the brainstem and amygdala, but potentially eliminating portions of 
either frontal or occipital cortex) if needed. 
Finally, we have not proposed to include 31P MRS in this initial, pilot study.  We are currently 
developing a 31P protocol and building the coil that will subsequently be used to acquire the 
31P MRS data. This part of the protocol would be added to the preliminary study provided 
that the coil is ready in the timeframe for initiation of this project. 
 
 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
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SAP_001.pdf: 
1 
 
Last modified: January 7, 2017 
 
 
 
 
 
 
 
 
Measuring the functional effects of BOTOX on the 
brainstem using MR Spectroscopy (MRS) and fMRI 
 
PI: Craig Stark, Ph.D., Professor 
 
 
 
 
 
 
 
320 Qureshey Research Lab 
Department of Neurobiology & Behavior 
University of California, Irvine 
Irvine, CA 92617 
(949) 824-4230 
cestark@uci.edu 
 
 
 


Measuring the effects of BOTOX on the brainstem using MR Spectroscopy (MRS) and fMRI 
  
2 
STATISTICAL ANALYSIS PLAN 
MRS Data Analyses 
Metabolite FIDs will be averaged within each task and processed using TARQUIN (v.4.3.6) 
software for spectral fitting. We chose a block design (vs. event-related) for its higher detection 
efficiency (at the cost of the inability to sort by individual trial performance or estimate HRF 
shape)75. Within-task dynamics will be investigated by a moving average windowing approach. 
The minimum temporal window will be determined by the SNR. The corresponding binned 
water unsuppressed spectra will be used as an internal reference for each condition. The 
acquired MRS spectra will be corrected for tissue type and T2 relaxation differences. 
Additionally, using TARQUIN we will obtain values for all metabolites mentioned in Preliminary 
results. These values will be collected to ensure that the changes in Glu and GABA signals are 
real and not due to a result of some other general effect on the MRS data (e.g., BOLD related 
change in signal). The water peak amplitude changes will be processed with a custom designed 
MATLAB script5.  
Functional MRI Data Analyses 
The functional MRI data will be pre-processed and analyzed using AFNI (Cox, 1996; 
afni.nimh.nih.gov) and in-house programs written in Matlab and Python. The fMRI data were 
first subjected to a traditional general linear model analysis using multiple regression in AFNI 
(Cox, 1996). The task vectors of interest will be individually modelled for each participant’s 
functional data utilizing a deconvolution approach built into AFNI's 3dDeconvolve (Ward, 2001).  
15 tent functions will be used to estimate the hemodynamic response to each condition.  The 
resultant fit coefficients (β coefficients) represent activity versus baseline for each condition of 
interest at a given time point in each voxel. The sum of the resultant fit coefficients over the 
bulk of the expected hemodynamic response (3-13.5 seconds after trial onset) will be taken as 
the model’s estimate of the response for each trial type and passed on to group-level analyses. 
The hypotheses will be primarily tested using the standard linear model. Brain activity for 
positive and negative emotions pre- and post-BOTOX injection will be compared in a 2x2 
ANOVA, with planned comparisons for follow-up. 
Resting state data will be detrended using 2nd order Legendre polynomials. The time 
series will be normalized to have zero mean and unit variance and the motion vectors and first 
derivatives will be regressed out of the signal. The first 6 principal components of the variance 
will be computed using CompCor (not including global signal) and regressed out of the signal 
(Behzadi et al., 2007). The data were then temporally bandpass filtered (0.009 to 0.08 Hz) and 
TRs with framewise displacement >0.5mm, as well as those one TR before and two TRs after, 
will be removed. 
Structural brain volumes will be calculated on the MP-RAGE for each participant using 
Freesurfer’s automatic software for volumetric measures (FreeSurfer 2016; Fischl et al. 2002). 
These volumes will then be normalized by dividing by total intracranial volume for each 
participant. T-tests for pre- vs. post- BOTOX injections can then be examined.