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Is it up there? - an MR spectroscopy study of frontal
lobes and non-primary-auditory temporal areas in
subjective bilateral and unilateral tinnitus
Joanna Wójcik
Bioimaging Research Center, World Hearing Center, Institute of Physiology and Pathology of Hearing
Bartosz Kochański
Bioimaging Research Center, World Hearing Center, Institute of Physiology and Pathology of Hearing
Katarzyna Cieśla ( kasia.j.ciesla@gmail.com )
Bioimaging Research Center, World Hearing Center, Institute of Physiology and Pathology of Hearing
Monika Lewandowska
Institute of Psychology, Faculty of Philosophy and Social Sciences, Nicolaus Copernicus University
Lucyna Karpiesz
Tinnitus Department, World Hearing Center, Institute of Physiology and Pathology of Hearing
Iwona Niedziałek
Tinnitus Department, World Hearing Center, Institute of Physiology and Pathology of Hearing
Danuta Raj-Koziak
Tinnitus Department, World Hearing Center, Institute of Physiology and Pathology of Hearing
Piotr Henryk Skarżyński
Institute of Sensory Organs
Tomasz Wolak
Bioimaging Research Center, World Hearing Center, Institute of Physiology and Pathology of Hearing
Article
Keywords: tinnitus, glutamate, magnetic resonance spectroscopy, frontal lobe, temporal lobe
Posted Date: February 28th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2594746/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Subjective tinnitus is a prevalent, though heterogeneous, condition whose pathophysiological
mechanisms are still under investigation. Based on animal models, changes in neurotransmission along
the auditory pathway have been suggested as co-occurring with tinnitus. It has not, however, been studied
whether such effects can also be found in other sites beyond the auditory cortex. Our MR spectroscopy
study is the rst one to measure composite levels of glutamate and glutamine (Glx; and other central
nervous system metabolites) in bilateral medial frontal and non-primary auditory temporal brain areas in
tinnitus. We studied two groups of participants with unilateral and bilateral tinnitus and a control group
without tinnitus, all three with a similar hearing prole. We found no metabolite level changes as related
to tinnitus status in neither region of interest, except for a tendency of an increased concentration of Glx
in the left frontal lobe in people with bilateral vs unilateral tinnitus. Slightly elevated depressive and
anxiety symptoms are also shown in participants with tinnitus, as compared to healthy individuals, with
the bilateral tinnitus group marginally more affected by the condition. We discuss the null effect in the
temporal lobes, as well as the role of frontal brain areas in chronic tinnitus, with respect to hearing loss,
attention mechanisms and psychological well-being. We furthermore elaborate on the design-related and
technical obstacles when using MR spectroscopy to elucidate the role of neurometabolites in tinnitus.
Introduction
Subjective tinnitus is an auditory percept (such as e.g. buzzing, ringing, humming), experienced despite
absence of any identiable external sound sources. Most authors suggest primary sensorineural hearing
loss as the main trigger for tinnitus occurrence, through the mechanism of peripheral
disruption/deafferentation of the neuronal activity1–7. This initial trigger is believed to then spark a series
of further changes up along the auditory pathway, including in the spontaneous or sound-induced neural
activity in regions spanning from the auditory nerve to the secondary auditory cortical areas, as shown in
animal models of tinnitus3,4,8,9 and in humans10–15.
At the molecular level, tinnitus is described in the framework of disrupted excitation-inhibition
homeostasis mainly mediated, respectively, by glutamate (Glu) and gamma-aminobutyric acid (GABA) at
several levels of the auditory pathway, including the cochlear nucleus, inferior colliculus, thalamus and
auditory cortex4,16. A series of studies have indicated changes in both these neurotransmitter systems,
including altered concentrations, tissue distribution, receptor anity and density, transporter function, in
rodents with behavioral signs of tinnitus17–23. These animal ndings led to initiating several drug trials
for tinnitus treatment, including of glutamate antagonists24,25. With several of these trials discontinued,
however, it has been emphasized that more research is still required regarding pathophysiology of
tinnitus26.
In addition to invasive methods, metabolite and neurotransmitter concentrations in the central nervous
system can also be studied using the non-invasive technique of 1H-MRS (proton magnetic resonance
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spectroscopy, hereafter MRS)27. In fact, Brozoski and colleagues28 applied MRS to show bidirectional
changes in GABA and glutamate levels in the cochlear nucleus, medial geniculate body and the auditory
cortex of rats with behavioral signs of tinnitus. The method has also been proposed as a tool to study
molecular aspects of tinnitus in humans29. Up until now, however, its implementation has been very
limited. To the best of our knowledge, there have only been three works investigating Glu or GABA levels
in tinnitus, with the authors only focused on the auditory cortex, as most probably inspired by animal
models16,30,31.
A critical challenge when studying neurotransmission in the auditory pathway, including in the primary
auditory cortex, is that changes due to tinnitus cannot be easily disentangled from those resulting from
hearing loss. This especially concerns animal models, with tinnitus induced by noise, physical or
chemical trauma, thus surely leading to severe hearing decits19,32,33. In humans there is a considerable
population of people with tinnitus and without a prominent hearing loss34–37. Nevertheless, there is also
evidence that tinnitus can stem from even a minor or a “hidden” hearing decit undetectable with
conventional pure tone audiometry6, 38–41.
A further signicant difference between human and animal models is that certain perceptual, cognitive
and emotional aspects of tinnitus can only be measured in humans36. At the same time, understanding
these aspects and the related mechanisms of tinnitus becoming a conscious chronic condition is also
most relevant for potential treatment development. In fact, increasingly more evidence shows that
following tinnitus generation the maintenance of tinnitus involves neuronal networks beyond the auditory
system. In particular, authors emphasize the role of prefrontal brain areas, as mediating conscious
perception of tinnitus6,42,43, its cognitive aspects44–48, and the co-existing distress36, 47–49.
Following this research, in the current MRS work in people with tinnitus, we included as regions of
interest: (a) temporal areas excluding the primary auditory cortex, in order to diminish the potential
confounding effect of hearing loss, and (b) regions encompassing medial frontal brain areas. Both these
areas are involved in all kinds of conscious auditory perception (such as during auditory hallucinations
and auditory imagery), as well as in auditory memory6, 50–52. We sought to investigate whether the
auditory models of neurotransmission in tinnitus can be expanded to include both these brain regions.
Participants also completed questionnaires assessing their well-being and subjective properties of
tinnitus.
Specically, we used single-voxel MRS and a standard clinical sequence to measure glutamate and
glutamine (Glx) composite levels, as a proxy for Glu. GABA signal quality was also evaluated, when
measured with our specic clinical sequence.
The regions of interest, the left and right medial frontal areas and bilateral superior/medial temporal
areas, were selected individually for each person in locations remote from bone, blood vessels and air
tissue, in order to obtain high-quality signals. Brain tissue segmentation was performed to ensure the
same content of white and gray matter across groups53–55.
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In order to address the issue of heterogeneity among the reported groups38, our study, beyond
investigating novel regions of interest, is also the rst one to apply a clear distinction between a subgroup
of participants that experienced tinnitus as bilateral and those whose tinnitus was perceived as unilateral.
Literature exploring the reasons for developing uni- or bilateral tinnitus is lacking, with several authors
discussing genetic factors underlying bilateral tinnitus56, involvement of higher order areas in the
emergence of unilateral tinnitus37, and hearing level asymmetries57. All participants had normal hearing,
except for several with a more severe hearing loss in middle/high frequency ranges, but the groups were
not different in that aspect.
As our main hypothesis, we expected to see a relationship between the status and laterality of tinnitus,
and higher glutamate levels (indicating increased neuronal excitation).
As part of general exploration, levels of several other metabolites were also measured in this study, i.e. N-
acetylaspartate (NAA), choline (Cho) and myoinositol (mI), all typically assessed in clinical brain MRS,
pertaining to their different functions in maintaining a healthy central nervous system (CNS), including
auditory system function16,27, 58–60.
Material And Methods
Participants
General demographics and exclusion criteria
Seventy seven (77) participants in total were included in the study, with 25 healthy subjects (group C) and
52 patients with tinnitus (24 with unilateral tinnitus, group TU, and 28 with bilateral tinnitus, group TB).
Screening included an interview, a detailed medical examination of the ear and an anatomical T1 or T2
MRI head scan, as well as a set of questionnaires. Participants with the following impairments or medical
history were excluded from the study: somatic tinnitus, acoustic tumors, brain tumors, stroke,
neurovascular conict, Meniere’s disease, cerebellopontine angle tumors, tumors of the internal meatus,
meningioma, astrocytoma, MELAS syndrome, history of surgeries of the auditory pathway or the brain,
history of neurological or psychiatric disorders (including clinical depression). The demographic data of
the participants is presented in Table1.
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Table 1
Demographic information.
Characteristic1C, N = 252TU, N = 242TB, N = 282p-value3
Age 0.7
Median (IQR) 42 (38–51) 48 (38–56) 44 (37–52)
Mean (SD) 45 (10) 46 (13) 44 (11)
Range 30–71 21–69 24–61
Sex
0.059
Female 10 / 25 (40%) 16 / 24 (67%) 10 / 28 (36%)
Male 15 / 25 (60%) 8 / 24 (33%) 18 / 28 (64%)
1SD - standard deviation, IQR - inter-quartile interval
2C - control, TU - tinnitus unilateral, TB - tinnitus bilateral For sex: n / N (%); n-subgroup count, N -
whole group count;
3For age: Kruskal-Wallis rank sum test; For sex: Pearson's Chi-squared test
The characteristics of tinnitus
The included fty-two (52) participants with tinnitus had been recruited from among the patients of the
Institute of Physiology and Pathology of Hearing. All had experienced tinnitus for at least 6 months
continually (considered
chronic
tinnitus). Unilateral tinnitus was mostly described by the participants as “I
hear it on the left/right side” (20 out of 24 perceived their tinnitus as left-lateralized and 4 as right-
lateralized). Five out of 24 presented clear one-ear dominance, while perceiving minor tinnitus in the other
ear. Bilateral tinnitus was described by the participants as “I hear it in the head” or “I hear it in both ears”.
Five out of 28 people reported that their tinnitus laterality was changing but generally affecting both ears.
Subjective experience of tinnitus was variable among the participants, and they characterized the sound
as tonal, hissing, buzzing or ringing, as well constant or changing in time, frequency and/or intensity.
None of the participants took part in any kind of therapy or training targeted at tinnitus prior to or during
the experiment.
Participants with tinnitus completed the following questionnaires referring to their tinnitus experience:
1. Tinnitus Handicap Inventory (THI) consisting of 25 questions describing the inuence of tinnitus on
everyday life. The scoring scale is from 0 to 100 points. The score 0–16 is interpreted as slight
inuence of tinnitus, 18–36 as mild, 38–56 as moderate, 58–76 as severe, and 78–100 as
catastrophic inuence61–63.
2. Tinnitus Functional Index (TFI) consisting of 25 questions describing how the respondent has been
feeling during the last week, with respect to tinnitus. The general score of TFI is on a scale of 0-100
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points. Tinnitus is considered a small problem when the scores are in the range between 0–18
points. For higher scores, the following interpretation is used: 18–42 points - a moderate problem,
42–65 points - a signicant problem, 65–100 points - a very signicant problem63–65.
3. The Polish Tinnitus Characteristics Inventory (the original Polish name: “Kwestionariusz
charakterystyki szumów usznych”) which is an in-house instrument developed by specialists of the
Institute of Physiology and Pathology of Hearing. The inventory measures, among others, on a VAS
scale, persistence of tinnitus (“Please, rate the persistence of your tinnitus on a scale from 1 (Mild) to
10 (Very persistent)”), awareness of tinnitus (“Assuming that the whole day (without sleep) is 100%,
please state on average the percentage of the day that you are aware of your tinnitus”), and time
since onset of tinnitus.
Hearing prole
The three included groups of participants (two tinnitus subgroups and the healthy controls) were not
different in terms of their hearing loss characteristics. Mean group hearing loss, for each frequency range
from 125 and 8000 Hz for all groups was within the normal range according to WHO66. Several
individuals in each group had some degree of hearing loss: 5 people in the control group, 2 people in the
TU group, and 4 people in the TB group, had a PTA (pure tone average, mean HL for 500–4000 Hz)
exceeding 25 dB in at least one ear. Figure1 shows mean group audiograms; individual PTA levels for
each participant are shown in Supplementary Fig.1. Two participants from the healthy control group did
not undergo the tonal audiometry test due to time constraints but had no history of hearing problems.
None of the participants was using any audiological devices (i.e. e.g. cochlear implants or hearing aids)
prior or during the study. All participants used spoken communication and had no diagnosed problems
with language development or speech understanding.
In order to better characterize the hearing prole of the participants, PTA values were compared across
groups separately for the right ear (or contralateral to tinnitus), the left ear (ipsilateral to tinnitus), the
better ear, the worse ear, and the hearing level asymmetry (difference between the ears). The results are
presented in Supplementary Table1 and show no signicant between-group differences. The three
groups were also not different with respect to their hearing levels at 8 kHz.
General psychological questionnaires
All participants completed two screening questionnaires (except for one person from the control group
and one person from the TU group) referring to their psychological functioning:
1. State-Trait Anxiety Inventory (STAI) measuring anxiety tendencies which includes 40 questions, 20
referring to anxiety as a state and 20 referring to anxiety as a trait. The respondent answers the
questions with respect to the extent that they refer to him/her behavior and personality. The
minimum row points that can be obtained in each scale is 20 and the maximum is 80. The scores
are summed up and converted to a sten scale. According to the generally accepted norms, a score
corresponding to 1st-3rd sten is considered
very low
or
low
, a score at the level of 4th-6th sten is
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considered
average
, and a score at the level of 7th-10th sten is considered
elevated, high
or
very
high
67,68.
2. Depression Assessment Questionnaire (DAQ, the original Polish name: “Kwestionariusz do Pomiaru
Depresji”) measuring depression tendencies using 75 statements. The respondent chooses one of 4
answers: 1) never, 2) sometimes, 3) often, 4) always or 1) very much, 2) signicantly, 3) slightly, 4)
not at all, with respect to how much a given statement refers to him/herself. The maximum overall
score is 240 (the minimum is 0). The scores are summed up and converted to a sten scale.
According to generally accepted norms, a score corresponding to 1st-3rd sten is considered
very low
or
low
, a score at the level of 4th-6th sten is considered
average
, and a score at the level of 7th-10th
sten is considered
elevated, high
or
very high
69.
Ethics statement
All study participants signed an informed consent to take part in the study and were not compensated for
participation. The study was approved by the Bioethical Committee of the Institute of Physiology and
Pathology of Hearing (number of approval KB.IFPS.13/2016 with changes KB.IFPS.29/2017) and
conformed to the Declaration of Helsinki from 2013.
MRI and MRS data collection
MRS data for the experiment had been collected in the years 2018–2020. All participants were scanned in
a 3T Siemens Prisma FIT scanner (Siemens Medical Systems, Erlangen, Germany) at the Bioimaging
Research Center of the Institute of Physiology and Pathology of Hearing, with a 20-channel receiver head-
coil. A single voxel spectroscopy (SVS) PRESS (Point-Resolved Spectroscopy Sequence) sequence was
applied for collection of MRS data from four locations in the brain: left temporal lobe, right temporal lobe,
left frontal lobe, right frontal lobe. The parameters were: voxel volume = 1.5 cm x 1.5 cm x 1.5 cm (3.75
cm3), TR (time of repetition) = 2000 ms, TE (time of echo) = 40 ms, TA (time of acquisition) = 4 min 16 s,
128 averages with 1024 time points and 1200 Hz bandwidth. For single voxel localization, an anatomical
T2-weighted scan was performed in three orthogonal sequences: coronal (TR = 5000 ms, TE = 98 ms, slice
thickness 4 mm, FOV 197 x 220), transverse (TR = 4000 ms, TE = 88 ms, slice thickness 4 mm, FOV 197 x
220) and sagittal (TR = 3000 ms, TE = 111 ms, slice thickness 4 mm, FOV 220 x 220), for the total
scanning time of 8 min.
An experienced MRI technician located 4 voxels corresponding to the 4 regions of interest (ROIs)
manually in every person, using the coronal, the sagittal and the transverse T2 plane images, in temporal
and frontal lobes. The locations were chosen to minimize the content of the cerebrospinal uid, blood
vessels, bones and air cavities. The size of the voxel (3.75 cm3) used in the study was optimized to t
various individual brain anatomies (i.e. head sizes and relative distances from brain structures) and at
the same time to maintain reasonable scan duration. Shimming of the MRS data was automatic. Figure2
depicts group-level brain coverage of the selected voxels and a model of white matter tracts crossing
through the selected regions of interest.
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Data analysis
MRS spectra
Selection of Metabolites
Raw data from each voxel in each participant was analyzed with LCModel76 version 6.3-1R. The analysis
of spectra focused on the following components: Glx, tNAA (total NAA), tCho (total Cho), mI and GABA.
Glx is a composite measure of glutamate (Glu) and glutamine (Gln), metabolites with a signicant
spectral overlap (Ramadan et al., 2013). Similarly, tNAA is a composite measure of N-acetylaspartate
(NAA) and N-acetylaspartylglutamate (NAAG), and tCho is composed of phosphocholine (PCh) and
glycerophosphocholine (GPC)27. Metabolite levels are further analyzed as ratios to tCr (total creatine)
levels, e.g. Glx/tCr.
Spectra quality control
The MR technician made sure during data collection that signal-to-noise ratios (SNR) for all of the spectra
exceeded the value of 3. Only spectra with a linewidth (FWHM) below 0.1 ppm remained, according to the
general MRS spectra quality recommendations77. Following further guidelines for the LCModel, spectra
with Cramér-Rao lower bounds of standard deviation (CRLB) above 15% were removed.
Statistical analysis of the MRS spectra
The levels of each metabolite were summarized and then one-way Welch’s ANOVA was applied to assess
differences between concentrations in the three studied groups of participants (healthy, unilateral tinnitus,
bilateral tinnitus) in each of the 4 brain regions separately. This was followed by planned post-hoc t-tests
to compare the measured metabolite levels between the groups. Normality was inspected for each
group’s results distribution, using the Shapiro-Wilk test.
Tissue segmentation
As part of further quality control, tissue segmentation was performed to white matter (WM), gray matter
(GM) and the cerebrospinal uid (CSF), using high-resolution 3D T1 images of the study subjects. Two
participants did not undergo this additional T1 exam and so the tissue segmentation was not performed.
Voxel dimensions from SVS MRS les metadata were used as binary masks on the T1 images which
were segmented in SPM12 (SPM12 Software - Statistical Parametric Mapping; www.ucl.ac.uk) to obtain
probabilistic maps of WM, GM and the CSF content, for each person and voxel individually, for 4
locations (left and right temporal lobe, left and right frontal lobe). The mean group content of the tissue
types was compared between the three groups using a Kruskal-Wallis test.
Statistical analysis of the questionnaire scores
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The scores of the THI, TFI, DQ, STAI questionnaires and scales of awareness, persistence and duration of
tinnitus were summarized for each group separately and the scores were compared between the groups
using a Kruskal-Wallis test, followed by Wilcoxon rank sum tests (if the omnibus test was signicant).
Severity levels based on THI and TFI scores were also analyzed using Fisher’s exact test.
Correlation analysis between PTA values, Glx/tCr levels, and
Chol/tCr levels
Concentrations of Glu31,78 and Cho16 in regions of interest including the auditory cortex have been shown
to correlate with hearing loss levels (and thus confounding to effect of tinnitus
per se
). We veried
whether this effect exists in our data by testing for Pearson correlation between Glx and Cho levels and
PTA values derived from an audiometric evaluation.
Tools for analysis
For all the described analyses we used the following software: base R79 and tidyverse80 packages for
statistical analyses, FID-A for le conversion81, gtsummary82 to create tables, ggbeeswarm83 and
ggsignif84 for visualization.
Results
Psychological and tinnitus questionnaires
Table2 and Fig.3 present the results of two tinnitus questionnaires applied in the two groups of patients
with tinnitus. There were no statistically signicant differences found between the scores in the two
groups in none of the questionnaires (p > 0.05, Wilcoxon rank sum test). The TFI mean group scores of 26
and 28 points, in TU and TB, respectively, indicated small or moderate levels of tinnitus severity63–65. As
for the severity level distribution, in 36% of TB participants tinnitus was signicantly severe or worse, as
compared to 16% in the TU group, but the difference was not statistically signicant (p > 0.05). Mean THI
scores of 28 and 32, in TU and TB, respectively, indicated mild inuence of tinnitus on everyday life.
Statistically signicantly, 50% of TB participants reported slight inuence of tinnitus and in 18% the
inuence was mild, whereas in the TU group the proportions were 25% and 58%, respectively (p = 0.023).
According to the Polish Tinnitus Characteristics Inventory, in the TB group the mean awareness of
tinnitus (during the day) was higher, as compared to the TU group, but the difference was not statistically
signicant (p > 0.05). There were no effects revealed for any inter-group differences with respect to the
duration and persistence of tinnitus.
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Table 2
Results of tinnitus questionnaires.
Characteristic1TU, N = 242TB, N = 282p-value3
TFI 0.8
Median (IQR) 24 (11–32) 20 (6–50)
Mean (SD) 26 (18) 28 (24)
Range 4–73 1–70
TFI - problem level 0.15
small 9 / 24 (38%) 13 / 28 (46%)
moderate 11 / 24 (46%) 5 / 28 (18%)
signicant 3 / 24 (12%) 8 / 28 (29%)
very signicant 1 / 24 (4.2%) 2 / 28 (7.1%)
THI 0.7
Median (IQR) 26 (19–32) 20 (13–54)
Mean (SD) 28 (14) 32 (29)
Range 4–60 0–92
THI - inuence level 0.023
slight 6 / 24 (25%) 14 / 28 (50%)
mild 14 / 24 (58%) 5 / 28 (18%)
moderate 2 / 24 (8.3%) 3 / 28 (11%)
severe 2 / 24 (8.3%) 3 / 28 (11%)
catastrophic 0 / 24 (0%) 3 / 28 (11%)
Persistence of tinnitus > 0.9
Median (IQR) 6.00 (4.38–7.25) 6.00 (3.00–8.00)
Mean (SD) 5.69 (1.95) 5.54 (2.77)
Range 1.00–9.00 1.00–9.00
Awareness of tinnitus
0.090
Median (IQR) 40 (20–50) 50 (40–72)
Mean (SD) 41 (25) 53 (31)
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Characteristic1TU, N = 242TB, N = 282p-value3
Range 10–100 1–100
Months since tinnitus onset 0.5
Median (IQR) 58 (34–120) 60 (36–127)
Mean (SD) 85 (76) 92 (70)
Range 15–264 13–270
1SD - standard deviation, IQR - inter-quartile interval
2 TU - tinnitus unilateral, TB - tinnitus bilateral; for TFI and THI levels: n / N (%); n-subgroup count, N -
whole group count; Fisher's exact test
3For continuous variables: Wilcoxon rank sum test; For categorical variables: Fisher’s exact test
Table3 and Fig.3 depict the results of the DAQ and STAI questionnaires in three study groups. The mean
results for both DAQ and STAI-trait questionnaires were in the range of 4–6 sten in all three groups which
indicated average levels of the reported depressive symptoms and anxiety, respectively. Nevertheless,
both TU and TB groups had statistically signicantly higher DAQ scores than the healthy control
participants (p = 0.017, p = 0.023). Also in the STAI-trait questionnaire, the scores were higher in the TB
group, as compared to the control group, at a tendency level (p = 0.063). Both for DAQ and STAI, only in
the control group there was no participant with scores beyond the 7th sten indicating elevated/high levels
of symptoms.
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Table 3
Results of psychological questionnaires.
Post-hoc tests
Characteristic1C, N = 252TU, N = 242TB, N = 282p-
value3
C vs.
TU4
C vs.
TB4
TU vs.
TB4
DAQ sten score 0.007 0.017 0.023 > 0.9
Median (IQR) 4.00 (2.75–
5.00) 5.00 (4.50–
6.50) 6.00 (4.00–
7.25)
Mean (SD) 3.75 (1.75) 5.48 (1.95) 5.57 (2.36)
Range 1.00–7.00 2.00–9.00 2.00–10.00
Missing 1 1 0
STAI - Trait sten
score
0.060
0.4
0.063
> 0.9
Median (IQR) 4.00 (2.00–
4.00) 4.00 (3.00–
5.00) 5.50 (2.75–
7.00)
Mean (SD) 3.33 (1.71) 4.52 (2.33) 5.14 (2.88)
Range 1.00–7.00 1.00–10.00 1.00–10.00
Missing 1 1 0
1SD - standard deviation, IQR - inter-quartile interval
2C - control, TU - tinnitus unilateral, TB - tinnitus bilateral
3Kruskal-Wallis rank sum test
4Wilcoxon rank sum test; Bonferroni correction
MRS spectra after quality control
For Glx, 294 observations remained ultimately, including 77 from the left frontal voxel, 76 from the right
frontal voxel, 70 from the left temporal voxel and 71 from the right temporal voxel. For GABA, the CRLB
values of all the individual metabolite spectra exceeded 15% and therefore were not analyzed further. A
summary of all the quality control values for Glx is presented in Supplementary Table2. The CRLB values
and the number of rejected observations for metabolites assessed as part of an exploratory analyses (mI,
tNAA, tCho) are presented in Supplementary Table3.
Results of tissue segmentation
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Tissue segmentation showed that all 4 voxels/regions of interest mainly consisted of white matter (61–
97% in frontal regions, 43–80% in temporal regions; see details in Supplementary Table4) and there were
no differences between the three groups with respect to the percentage content of different types of
tissue (p > 0.05).
MRS results
Table4 and Fig.4 show levels of Glx/tCr in three groups separately. ANOVA revealed a tendency for a
difference in levels in the left frontal ROI (p = 0.055) between three study groups. Results of planned post-
hoc tests showed a tendency for increased Glx/tCr concentration in the TB group, as compared to the TU
group in the left frontal ROI (p = 0.058). As depicted, no statistically signicant differences were found
between the groups in the temporal ROIs and the right frontal ROI (p > 0.05). There was also no difference
in Glx levels revealed between the right and the left temporal ROIs in neither group (p > 0.05). The results
for tNAA, tCho, mI showed no statistically signicant differences between the groups either and have
been presented in Supplementary Fig.2 and Supplementary Tables5–7.
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Table 4
Glx/tCr levels in four regions of interest compared across groups
Descriptive statistics Welch ANOVA Post-hoc tests
Region1C, N =
252
TU, N
= 242
TB, N =
282
F p df1 df2 C
vs.
TU3
C
vs.
TB3
TU
vs.
TB3
Left frontal 3.1
0.055
2.00 49.1 >
0.9 0.3
0.058
Median
(IQR) 1.96
(1.69–
2.09)
1.78
(1.72–
2.09)
2.04
(1.87–
2.26)
Mean (SD) 1.94
(0.30) 1.88
(0.29) 2.08
(0.31)
Range 1.53–
2.60 1.49–
2.37 1.56–
2.81
S-W test 0.16 0.01 0.77
Missing 0 0 0
Right frontal 1.4 0.3 2.00 45.9 >
0.9 0.3 0.8
Median
(IQR) 1.86
(1.81–
1.98)
1.92
(1.70–
2.00)
1.97
(1.76–
2.28)
Mean (SD) 1.89
(0.20) 1.91
(0.29) 2.02
(0.37)
Range 1.58–
2.43 1.51–
2.74 1.23–
2.70
S-W test 0.18 0.04 0.43
Missing 0 1 0
Left/Ipsi
temporal 0.78 0.5 2.00 44.5 >
0.9 0.8 0.9
Median
(IQR) 2.44
(2.15–
2.68)
2.45
(2.11–
2.70)
2.26
(2.09–
2.43)
Mean (SD) 2.41
(0.38) 2.40
(0.36) 2.29
(0.35)
Range 1.56–
3.17 1.86–
3.15 1.68–
3.25
S-W test 0.98 0.46 0.42
Page 15/32
Descriptive statistics Welch ANOVA Post-hoc tests
Missing 1 2 4
Right/Contra
temporal 0.03 > 0.9 2.00 45.1 >
0.9 >
0.9 > 0.9
Median
(IQR) 2.29
(2.08–
2.70)
2.27
(2.12–
2.49)
2.35
(2.15–
2.43)
Mean (SD) 2.36
(0.41) 2.34
(0.35) 2.35
(0.42)
Range 1.68–
3.09 1.64–
3.08 1.64–
3.50
S-W test 0.43 0.47 0.01
Missing 1 3 2
1SD - standard deviation, IQR - inter-quartile interval, S-W test - Shapiro-Wilk test of normality result (p-
value), Ipsi – side ipsilateral to the perceived unilateral tinnitus, Contra – side contralateral to the
perceived unilateral tinnitus,
2C - control, TU - tinnitus unilateral, TB - tinnitus bilateral
3Welch t test; Bonferroni correction
Correlation analysis between PTA and Glx/tCr levels, PTA
and Chol/tCr
There was no statistically signicant correlation revealed between the hearing levels (PTA) of the
participants and the levels of Glx/tCr and Chol/tCr (as measured by Spearman’s ρ correlation test).The
results are presented in Supplementary Figs.3–4.
Discussion
Motivation and summary of the results
Magnetic resonance spectroscopy (MRS) remains a valuable non-invasive technique to study tinnitus.
The exact pathomechanisms and the underlying neuronal correlates of the condition are still not fully
understood. Our main aim was to expand the existing limited knowledge regarding the potential
involvement of glutamate/glutamine levels (basic excitatory neurotransmitter in CNS) and other
metabolites in the occurrence of subjective tinnitus. To the best of our knowledge, our work is the second
one, following Isler and colleagues31, that measured glutamate concentration with MRS specically in
people with tinnitus and a control group, and the rst one to investigate metabolite levels in frontal and
Page 16/32
non-primary auditory temporal brain areas in unilateral vs bilateral tinnitus subgroups. Our cohort of 52
tinnitus patients is the largest examined thus far with MRS and psychological questionnaires.
We showed a tendency of increased Glx/tCr levels in the left frontal brain area (but not temporal brain
regions) in people with bilateral tinnitus, as compared to those who perceived their tinnitus as unilateral.
At the same time, the presence of tinnitus
per se
(i.e. compared to the control) was not associated with
different levels of metabolites in either region of interest. This lack of strong effects might indicate
general health of the CNS in people with tinnitus but further research is needed.
The average scores in the screening depression and anxiety questionnaires were comparable to the
general population’s average in both tinnitus subgroups (and in the healthy control group), and the
participants reported the effects of their tinnitus on everyday life as mild to moderate. Nevertheless,
tinnitus was related to slightly elevated affective symptoms, and the well-being of the participants who
experienced their tinnitus as bilateral vs unilateral might have been more affected by the condition
(details are discussed further in the text).
Frontal brain regions in tinnitus: attention and the noise-canceling
mechanism
Showing higher relative concentrations of Glx in the frontal brain regions in participants with bilateral
tinnitus is in line with the growing number of studies indicating involvement of these areas in tinnitus
pathophysiology6,85, including in white matter tracts35, 86–88. Specically, the CC, cingulum and ACR
bers crossing our frontal regions of interest6,16,89 connect prefrontal and auditory regions. Distorted
communication between these two brain regions has been suggested as contributing to tinnitus
pathophysiology (see also work by Chen and colleagues86 who describe similar effects in functional
connectivity in tinnitus, as measured with resting state functional magnetic resonance imaging). The role
of frontal regions has been discussed in the context of several specic mechanisms related to tinnitus.
One line of research postulates that subsequent to peripheral auditory damage, the maintained
awareness of tinnitus is due to abnormal engagement of cognitive control and impaired attention-
switching (frontal lobe functions), mediated by the limbic system39, 43–46,87. According to another yet
related hypothesis the prefrontal cortex is part of a noise canceling/inhibition system (which also
includes the thalamic reticular nucleus adjacent to the auditory thalamus, MGB) whose permanent failure
leads to tinnitus becoming chronic6,39,42,44,90. Specically in our study, Glx levels were higher in bilateral
as compared to unilateral tinnitus in the
left
prefrontal region. As proposed by Palmiero and colleagues91,
this region has been shown to be engaged in executive control and specically in inhibition of negative
(vs positive) distractors. We can thus speculate that the relationship between Glx levels and tinnitus
laterality is due to more ecient attention control and/or noise canceling (inhibition) system in unilateral
tinnitus, which might have also prevented development of a bilateral tinnitus percept (indeed see an
indication for impaired selective attention in bilateral tinnitus in works by Sharma et al.92 and Bartnik et
al.93). In other words, the perceived laterality of tinnitus may be mediated by the frontal lobe. In our study
Page 17/32
there were no differences shown in hearing level asymmetry between the two tinnitus groups that might
otherwise potentially account for the perceived tinnitus laterality (cf. Genitsaridi et al.57).
Frontal brain regions in tinnitus: psychological well-being
In addition to potentially different attention mechanisms, one could also consider bilateral tinnitus a
serious chronic stress inducer85,94, which might have led to higher levels of Glx than in unilateral tinnitus.
Exposure to chronic stress has been shown, in a comprehensive review, to cause excessive glutamate
levels in the frontal brain regions before leading to neuronal atrophy95. Changes in glutamate levels in the
medial prefrontal cortex have also been suggested to reect stress adaptation96.
In fact, beyond metabolite level changes, we also found some effects related to the psychological
functioning of the individuals with tinnitus. First of all, both people with unilateral and bilateral tinnitus
showed higher average levels of depressive symptoms than the healthy controls. Although the mean
levels were still in the range of the general healthy population average, about 25% individuals in both
tinnitus groups had scores showing high symptom levels. With respect to the anxiety screening tool,
participants with tinnitus perceived on both sides had higher levels of the symptoms than the control
group (at a tendency level). Furthermore, although these effects were not statistically signicant, in the
bilateral vs unilateral group there were more participants assessing the perceived tinnitus-related
handicap as high and this group was also more aware of their tinnitus in their daily life.
A number of behavioral studies has shown a bidirectional relationship between tinnitus and
psychological challenges, such as distress, depression and anxiety48,49, 97–99. Most of these studies
included participants with the bilateral type of tinnitus (which is more prevalent) or participants with
mixed lateralities. Although the data is scarce, few epidemiological studies comparing the two types of
tinnitus also indicated more annoyance57,92, tinnitus handicap100 (although see work by Song et al.37
who found a reverse effect) and depressive symptoms100 when tinnitus was perceived bilaterally.
In addition, MRS studies have shown a relationship between psychological functioning and glutamate
levels in frontal brain regions in patient populations with schizophrenia and/or depressive disorders,
including the prefrontal cortex, as well as the CC and ACR white matter tracts, both included in our frontal
regions of interest (see e.g. a review by Luttenbacher and colleagues101).
In sum, although subjects with
diagnosed
psychological and psychiatric diseases were excluded during
recruitment for the study in order to avoid the potential confounding effects of the disorders (and
possibly the related distress), the relationship between tinnitus and psychological status still remained.
This is indicated by slightly elevated affective symptoms in two screening questionnaires (DAQ and STAI;
cf. Ivansic et al.97). At the same time, the exact connection between MRS results and those based on the
subjective questionnaires (here: both higher Glx in the left frontal cortex and slightly more prevalent
psychological symptoms in bilateral tinnitus), has to be studied further in more sizable populations.
Page 18/32
Null effect in the temporal lobe excluding the auditory cortex
We could not establish any tinnitus-related alterations in metabolite levels in higher-order temporal
regions in vicinity though excluding the primary auditory cortex. We also found no relationship between
hearing levels and metabolite levels in the selected temporal regions (and all groups were matched in that
respect). It is therefore possible that physiological and metabolic/neurotransmitter changes
accompanying tinnitus are only present or are more profound in the auditory cortex (including the primary
auditory cortex adjacent to and including the Heschl gyrus) as indicated in rich animal and human
research16–23, 28,30,31,102,103. Although it still remains unclear whether the reported effects are due to
tinnitus
per se
or the accompanying hearing loss, even if non-severe (as in e.g. Isler et al.31 and Yoo et
al.88).
The temporal region of interest (in the medial/superior temporal lobe) in our study included white matter
tracts, such as the uncinate fascicle (UF), the fronto-occipital fascicle (FOF), and the inferior and middle
longitudinal fascicles (ILF, MLF). These tracts have been shown to be involved in episodic memory,
language content processing, and learning; in MLF and FOF through their connections with the Heschl
gyrus and in UF and FOF through their connections to the prefrontal lobe104–107. Also the gray matter
functional regions of the superior and medial temporal lobes have been described in the context of
language and memory functions108. With respect to tinnitus specically, the mismatch between auditory
memory of sounds and the distorted signal derived from the damaged peripheral auditory system has
been hypothesized to lead to the tinnitus percept and involve superior/medial temporal lobes6. We
speculate that the reason for the null effect of tinnitus in higher-level temporal regions in our study might
be due to the relatively long duration since tinnitus onset in all participants, during which signicant
neuroplastic changes might have already happened with remaining effects only visible in the frontal
brain areas (and possibly the limbic system). Another potential might be the specic location of the
region of interest which also mainly included white matter tissue. Further studies are required.
Tinnitus heterogeneity and participants’ inclusion criteria
Despite the high prevalence of tinnitus worldwide (an estimated 10–25% of the population worldwide),
there no one-size-ts-all treatment available at the moment25,93, 109–112. Tinnitus poses a signicant
research challenge as it is an extremely heterogeneous condition, both with respect to its origin, possible
manifestations and comorbidity with hearing loss.
In the few published MRS studies in people with tinnitus, the authors included groups either with mixed
tinnitus lateralities or various hearing loss levels16,30,31. Furthermore, studies differed between each other
in the mean age and mean hearing levels of participants (from almost normal PTA values to severe
hearing loss), mean time since tinnitus onset and tinnitus handicap scores. All these factors possibly
contributed to the variability in the results.
Page 19/32
In our study, with the aim to examine the relationship between glutamate (and other metabolites) levels
and tinnitus
per se
, we applied several exclusion criteria with respect to comorbid medical conditions, as
well as diagnosed psychological and psychiatric disorders. To avoid further confounding effects, the
three study groups were also similar with respect to age and PTA values (although we failed to exclude
hearing loss cases completely). Both these factors have been found to inuence metabolite levels in
CNS78, 113–116. We were also the rst ones in human MRS research, to divide the groups based on tinnitus
status and laterality. In future more sizable studies, further subtypes of tinnitus could be distinguished
(e.g. based on the perceived type of sound, loudness, distress levels, duration of tinnitus, awareness,
participation in tinnitus therapy/training, depression and anxiety levels, etc.), which might enhance the
effect of Glu level differences in various brain regions. Especially the bilateral tinnitus group might benet
from subtyping. In our data, as an example, both the scores in the psychological questionnaires and the
relative metabolite levels were consistently more sparsely distributed in this subgroup than in the
unilateral tinnitus group or the healthy subjects. Further improvement of methodology can include an
extended broad-band and high-resolution tonal audiometry test, to control the levels of hearing even more
precisely41,117,118.
MRS data quality control
Changes in brain metabolite levels, as measured with MRS, might be very subtle and prone to numerous
confounding effects intrinsic to MR imaging, e.g. signal originating from tissues of no interest, subject’s
movement or magnetic eld inhomogeneity119. In our study, before applying any statistical analyses, we
therefore performed data quality check (see
Methods
regarding SNR and FWHM of the acquired
metabolite spectra, CRLB of the estimated metabolite levels, WM/GM tissue composition). We also used
a PRESS sequence with a TE value optimized to measure Glx concentration120. In addition, the signal
was collected from relatively small localized ROIs placed possibly far from the skull and the sinuses to
minimize signal distortions (a specic challenge in MRS studies of the prefrontal cortex and the temporal
lobes77). The resulting ROIs contained a considerable amount of white matter (WM) tissue53,55, in which
physiology, as recent data shows, glutamate might play a signicant role, supporting myelination and
glial receptor function which then enhances axon potential initiation and propagation of impulses121–125.
At the same time, WM and GM tissue has been shown to affect the MRS signal differently53–55. We were
unable to report levels of GABA in the same regions of interest as for Glx and other metabolites, although
it might have provided a broader overview of the excitation-inhibition mechanisms in tinnitus. The reason
was, nevertheless, unreliable signal quality afforded by the available clinical sequence (PRESS; as also
shown e.g. in Baeshen et al.126) and lack of availability of a dedicated commercial MRS sequence (such
as e.g. MEGA PRESS).
Challenges of the MRS technique and future directions
Page 20/32
Aside from the discussed between-group differences, there is also considerable diversity among the
applied MRS methods, both in terms of the scanning sequences and data analysis. As an example, in
each of the three published MRS studies in tinnitus the authors applied a different MRS sequence
available for 3T MR scanners (STEAM, MEGA-PRESS, 2D-JPRESS, PRESS), used different software for
data analysis (LCModel, Gannet, ProFit) and applied various criteria for data quality. It has been shown
that these aspects contribute signicantly to the MRS measurements124,127. Future studies might benet
from more replicability, with the ongoing efforts to develop and establish uniform MRS research
protocols, including optimal scanning sequences and quality measures; with several expert consensuses
and recommendations for advanced MRS applications issued recently128–130. Beyond the mentioned
issues, some inconsistencies also exist considering the size of the region of interest in single-voxel
spectroscopy. Some authors opt for using large voxels for local metabolite measurements (e.g. in tinnitus
MRS studies: Isler et al.31; Sedley et al.16) that, however, do not warrant high regional specicity. Others
use voxels of smaller sizes, which approach nevertheless requires signal-to-noise ratio to be improved,
e.g. by repeated measurements (e.g. our study and Cacace et al.30). In addition, the reliance on a single
voxel (as in SVS MRS) yields its placement a crucial part of the design and can become a source of
variability in the published results131. The emerging whole-brain MRS (MRS imaging, MRSI), with data
collection from multiple small regions of interest simultaneously, can become an alternative to SVS MRS
in that respect. However, availability of vendor-provided advanced sequences for MRSI is limited as of yet
and the technology requires considerably more expertise in data quality assessment and
interpretation129,132. Finally, recent advances in MRS sequence development including implementation in
several commercial devices133, new signal processing methods134, real-time movement correction
algorithms135 and application of high-eld 7T MR scanners might all improve the reliability and
reproducibility of MRS measurements in the future.
Conclusion
In conclusion, we show no relationship between tinnitus status and levels of Glx, tNAA, tCho and mI levels
in the non-primary auditory temporal, and frontal brain regions. Nevertheless, our results indicate that
glutamate concentration in the left frontal lobe may play a different role in the development and
maintenance of bilateral, as compared to unilateral subjective tinnitus. The potential mechanisms may
be related to the functioning of the frontal-limbic-auditory noise canceling/inhibition system, attention
control as well as psychological aspects of tinnitus. Further research is still necessary to explore the
relationship between the tinnitus percept and the neurotransmitter systems in the brain. Emerging
methodological advances in MRS may facilitate the process.
Declarations
Data Availability
Page 21/32
The datasets generated and/or analyzed during the current study are not publicly available due to the
privacy issues of clinical data but are available from the corresponding author on reasonable request.
CRediT authorship contribution statement
J.W.: Conceptualization, Data curation, Investigation, Methodology, Project administration, Validation,
Writing - original draft, and Writing - review & editing. B.K.: Data curation, Formal analysis, Methodology,
Software, Validation, Visualization, Writing - original draft, and Writing - review & editing. K.C.:
Conceptualization, Methodology, Supervision, Validation, Writing - original draft, and Writing - review &
editing. M.L.: Conceptualization, Funding acquisition, Investigation, and Methodology. L.K.: Resources.
I.N.: Resources. D.R.-K.: Resources. P.H.S.: Resources and Supervision. T.W.: Data curation, Formal
analysis, Methodology, Software, Supervision, and Visualization.
Additional information
The authors declare no competing interests.
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Figures
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Figure 1
Average audiograms for each group with standard deviations marked as vertical error bars. The gray
rectangle represents normal hearing according to WHO; left = left ear, right = right ear, tinnitus ipsi - the ear
corresponding to the side of tinnitus in the TU group; for 4 TU participants that were experiencing tinnitus
on the right side, right ear hearing levels were combined with left-ear hearing levels of other participants
as
tinnitus ipsi
.
Figure 2
(A) Mean group locations of the 4 selected regions of interest (ROIs) - left frontal (red), right frontal (blue),
left temporal (green), right temporal (purple) - shown in MNI space over Automated Anatomical Labeling
atlas parcellation. Voxel brightness encodes the overlap of the voxel locations across subjects. Semi-
Page 31/32
transparent colors represent atlas parcellation. Image prepared in MRIcroGL, using Colin 27 brain as a
template70,71. Based on Automated Anatomical Labeling atlas, temporal ROIs included bilateral posterior
superior and medial temporal cortex, and the frontal ROIs contained bilateral frontal poles/anterior
cingulate gyri (les with mean group ROI masks are included in
Supplementary Materials
). (B)
Reconstruction of white matter tracts in 4 ROIs in an example single-subject brain, using FACT – Fiber
Assignment by Continuous Tracking72. A Julich DTI atlas73–75 was applied to identify white matter tracts
in each ROI. Frontal ROIs contained parts of forceps minor (FM), anterior corona radiata (ACR), cingulum
and corpus callosum (CC). Temporal ROIs uncinate fascicle (UF), fronto-occipital fascicle (FOF), inferior
longitudinal fascicle (ILF), middle longitudinal fascicle (MLF)
(https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Atlases).
Figure 3
Scores in the tinnitus questionnaires (upper row) and the depression/anxiety questionnaires (bottom
row). Groups are color coded. Individual subjects are depicted as points in the violin plots. White dots
represent group means while middle horizontal bars on box plots represent medians. TB - bilateral
tinnitus, TU - unilateral tinnitus, C – control, TFI - Tinnitus Functional Index, THI - Tinnitus Handicap Index,
DAQ - Depression Assessment Questionnaire, STAI - State-Trait Anxiety Inventory; awareness, duration
and persistence were assessed on a VAS scale. For TFI and THI raw scores are represented. DAQ and
Page 32/32
STAI results are presented on a sten scale. * - p < 0.05, · - p < 0.1, † - raw score difference not signicant,
categorized results difference signicant.
Figure 4
Glx/tCr levels in the four ROIs. Individual subjects are depicted as points in the violin plots. White dots
represent group means while middle horizontal bars on box plots represent medians. TB - bilateral
tinnitus, TU - unilateral tinnitus, C - control; Ipsi - the side corresponding to the side of tinnitus in the TU
group (for 4 TU participants that were experiencing tinnitus on the right side, the sides were switched),
Contra – side opposite to tinnitus laterality. · p < 0.1
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
File1LF.nii.nii
File2RF.nii.nii
File3LT.nii.nii
File4RT.nii.nii
Supplementarymaterial.docx
