The association between hearing loss and tinnitus corroborates earlier research and is a long standing finding. Prevalence of hearing loss increases with age (Davis, 1995), hearing loss increases the risk for developing tinnitus (Hoffman and Reed, 2004), and on a population level there is a linear increase in tinnitus annoyance with increasing age (Davis and El Rafaie, 2000; Andersson et al., 2005). Studies indicate that 70–80% of tinnitus patients have significant hearing difficulties (Henry et al., 2005; Hesse and Schaaf, 2015). Other studies also reported the positive link between higher hearing loss and higher tinnitus distress (e.g., Goebel and Hiller, 1999; Davis and El Rafaie, 2000; Stobik et al., 2003). For example, Savastano (2008) investigated 520 persons suffering from tinnitus and compared tinnitus patients with and without hearing loss. The author found that subjective discomfort is higher in the presence of hearing loss than in the case of normal hearing (according to Bureau International D'Audiophonologie pure tone average for 500 Hz, 1, 2, and 4 kHz < 20 dB). Among subjects with normal hearing, the level of disturbance was mostly in the moderate range, whereas among subjects with hearing loss, the level of disturbance was mostly elevated. Savastano concluded that the presence of hearing loss increases the complaint of tinnitus considerably, even if the hearing deficit is not severe.

Similarly, the correlation of tinnitus annoyance with otological symptoms reported here corroborates earlier research. Goebel and Hiller (2007) found a strong association between otological conditions and the development of high annoyance: subjects with additional hearing loss and dizziness/vertigo reported both higher loudness and higher annoyance. When subjects with high versus low annoyance were compared, the following odds ratios (OR) were found: hearing loss OR = 5.64 and dizziness/vertigo OR = 3.76. Hallam and Stephens (1985) found that tinnitus patients who complained of dizziness also suffered from higher emotional distress. Both Erlandsson et al. (1992) and Langenbach et al. (2005) observed a worsening of mood and tinnitus symptoms when tinnitus was accompanied by vertigo. The latter study also confirms our finding that loudness of the tinnitus perceived in the right ear correlated with higher tinnitus annoyance at the first appointment. Thus, the sound perceived in the right ear has a stronger impact on the associated emotional processes. This mechanism is not well understood and should be investigated in future studies.

The association between high tinnitus annoyance and poor maskability was also reported in several studies. Goebel and Hiller (1999) reported higher tinnitus annoyance when maskability was poor. Maskability of tinnitus at admission to CBT was a predictor of tinnitus-related distress at a 5-year followup (Andersson et al., 2001). Stobik et al. (2005) compared patients with low/moderate and severe/very severe tinnitus distress and also found that patients with severe/very severe tinnitus reported greater difficulty to mask their tinnitus with background sounds.

The relationship between tinnitus and emotional distress or psychiatric problems has long been recognized and is well documented, at least in the help-seeking group (Harrop-Griffiths et al., 1987; Dobie et al., 1992; Andersson, 2002). Sixty-three to seventy-seven percent of tinnitus inpatients have at least one psychiatric diagnosis (mostly mood or anxiety disorder; Kaldo, 2008). The prevalence of concurrent depression or mood disorders ranges between 39 and 60%, whereas the lifetime prevalence amounts to 62–78% (Andersson, 2002). Other psychological causes of distress associated with tinnitus include anxiety, depression, irritability, anger, and insomnia (Wilson et al., 1991). Approximately half of the patients with tinnitus without severe hearing impairment also suffer from psychiatric disorders, the most frequent being anxiety disorders and mood disorders (Zöger et al., 2001). Belli et al. (2008) applied the Structured Clinical Interview for DSM-III-R at 90 acute tinnitus patients and found that 24.4% had at least one axis-I psychiatric diagnosis. The most prevalent disorders were anxiety, somatoform and mood disorders. Only 6% of controls without tinnitus had at least one axis-I psychiatric diagnosis. Depression, sleep disorders and difficulties in concentration were significant predictors of tinnitus annoyance (Scott et al., 1990). Erlandsson and Hallberg (2000) investigated in 122 tinnitus inpatients which factors predict quality of life, and found that impaired concentration, feeling depressed and perceived negative attitudes were the most significant predictors and explained most of the variance in quality of life. In the study from Langenbach et al. (2005) on acute tinnitus, insomnia attributed to tinnitus was the best predictor and accounted for 34% of the variance of tinnitus distress 6 months after tinnitus onset. Anxiety in the acute phase accounted for 30% of variance of tinnitus distress, while life satisfaction and somatic complaints accounted together for 41% of the variance.

We conclude that our results of predicting tinnitus annoyance corroborate previous results. Taking Jastreboff's neurophysiological model of tinnitus into account, the negative impact of these symptoms on developing tinnitus annoyance is quite obvious. As the frequency of the individually perceived tinnitus is very likely to be in the range with the highest hearing loss (Henry et al., 1999; Noreña et al., 2002), the masking of tinnitus with background sounds is, in the case of hearing loss, not possible any more. Habituation inhibits tolerance to a stimulus because of its unpredictability. This is possibly why the variability of tinnitus during physical stress attracts attention to the phantom sound, making it difficult to habituate, which in turn leads to higher annoyance. If a person experiences tinnitus onset simultaneously with dizziness, the fear evoked by dizziness will be associated with the noise according to the principles of classical conditioning. Whenever tinnitus is perceived as a danger, no habituation can be achieved. This risk certainly gets higher, due to the belief that hearing loss or dizziness is caused by tinnitus, which is something that many of our patients reported. In the same direction, emotional distress or psychiatric problems are generally regarded as factors hindering habituation.

#### Predictors of Treatment Outcome

Inspecting changes in tinnitus annoyance in response to treatment, data from patients with moderate, severe and very severe tinnitus annoyance reached high effect sizes. But if we consider only a clinically significant change, JITT displays the strongest effects in patients with severe tinnitus (grade 3). Although tinnitus annoyance is significantly reduced in patients with very severe tinnitus (grade 4), the 5-day treatment is not sufficient to lead to a clinically significant change in this group. Perhaps this group of patients needs an extended duration of JITT or some other outpatient therapeutic approaches. Another possibility would be intensive inpatient care, which however removes patients from their daily routine. Also other researchers found that patients with high tinnitus annoyance at baseline were more often non-responders (Rübler, 1997; Frenzel, 1998) in outpatient setting.

Demographic, tinnitus and strain variables explained only 12.8% of the variance of the change in tinnitus annoyance from T0 to T4. The only predictors for reduction of tinnitus-related distress were "sick leave 6 months before the treatment onset" and "tinnitus annoyance at T0." Patients who were on sick leave before the treatment or with high tinnitus annoyance at T0 showed less improvement in tinnitus annoyance from treatment begin to the final follow-up. We did not inquire further why the patients were on sick leave, but they most likely experienced somatic/psychological symptoms to such an extent that they were unable to continue with their daily activities. The 5-day treatment was perhaps too short for this subgroup. It is yet unclear if they would benefit from a longer treatment duration or a combination of treatments as suggested below. Including "early change" as a predictor allowed to explain 27.4% of the variance, i.e., considerably more. Early change has generally proven to be a strong predictor in psychotherapy and CBT in particular (Schibbye et al., 2014). We did not include this variable in the first regression analysis, because it already requires knowledge about the responsiveness of a patient which was not given at T0. Nevertheless, it constitutes an early and easy calculable indicator who will respond to treatment and who is more resilient. Providing knowledge about early change to therapists could result in more effective treatment (Lambert and Ogles, 2003). Measuring early change seems well constituted for psychiatric disorders (Schibbye et al., 2014; Koffmann, 2017 for recent references), but it is less known in tinnitus research. We propose to integrate such measures in clinical settings for tinnitus treatment. As examples, the Clinical Outcomes in Routine Evaluation questionnaire (CORE, Evans et al., 2000) and the Outcome Questionnaire-45 (OQ-45; Lambert et al., 2004) in combination with the TQ as a disorder specific measure provide validated tools.

Even though the low predictability for treatment success is unsatisfactory, previous studies similarly failed to predict therapy outcome. Rief et al. (2005) found that age and illness duration had only marginal associations with treatment success. Baseline scores of tinnitus annoyance (TQ), gender of the patient or comorbidity with mental disorders were not significant predictors of outpatient psychological treatment. Neither duration of tinnitus nor the level of sleep disturbance, comorbid psychopathology, hearing problems, or experienced stress level affected the outcome of outpatient treatment (Kröner-Herwig et al., 2006). On the other hand, patients with high tinnitus annoyance and comorbid psychopathology at baseline were more often non-responders (Rübler, 1997; Frenzel, 1998). Goebel (2008) conducted a 15-year followup after inpatient tinnitus therapy on 271 tinnitus patients. Noise-induced tinnitus, gender and comorbid psychopathology explained 7.6% of the variance in tinnitus annoyance. Male tinnitus sufferers as well as patients with noise-induced tinnitus and high psychopathology reported higher tinnitus annoyance 15 years after the treatment.

Consequently, it appears that there is only little agreement on what can predict treatment outcome, but in no study was predictability good. The complexity of these processes was again stressed by Caffier et al. (2006). In their study, severely affected tinnitus sufferers showed clear improvements in TQ scores without any age-specific differences. In comparison, the groups of younger and older patients with mild tinnitus severity showed higher reductions in TQ scores in comparison to middle-aged patients between 46 and 56 years. Regarding preexisting tinnitus duration, patients with mild tinnitus annoyance demonstrated a particularly strong reduction of annoyance when the tinnitus lasted for less than 1 year. In contrast, in severely affected tinnitus sufferers, preexisting tinnitus duration did not seem to play a role for treatment success.

These results indicate that in order to predict treatment success by patient characteristics, we have to make subgroups and investigate which combinations of subgroup characteristics lead to better/poorer treatment success. Therefore, the next milestone in tinnitus research should be to update large data registries, into which standardized variables can be entered by independent tinnitus researchers. A tinnitus database has already been established by the Tinnitus Research Initiative (http:// www.tinnitusresearch.org/index.php/for-researchers/tinnitusdatabase) and will be improved and enlarged by the TINNET European research network funded by the COST program (http://tinnet.tinnitusresearch.net/). Such a central database will enable the specification of subgroups of tinnitus patients worldwide, making it more possible to develop individually tailored treatments for tinnitus patients.

# LIMITATIONS

Due to the lack of a control group receiving a different treatment, we cannot indicate how effective the present approach is compared to other possible treatments or, in the worst case, if it is due to a placebo effect or the mere passing of time. The interdisciplinary treatment comprised several modules, but whether one of them or a specific combination contributed to the reported effects remains to be tested. Future studies might adopt a dismantling approach, leaving out potentially redundant treatment components. Furthermore, cost-effectiveness studies and equivalence trials should be performed.

Even though the results of this study speak in favor of JITT, it must be noted that 35% of the tinnitus patients seen at the ENT department did not accept "habituation to tinnitus" as an objective of the intervention. Therefore, specific treatment approaches adapted to such patients should also be developed. We propose a combination of two therapeutic methods, one addressing tinnitus distress and the other the symptom itself. As an example for the latter, some evidence was presented that tailor-made notched music training reduces tinnitus loudness (Stein et al., 2016). Neurophysiological models (Jastreboff et al., 1996) suggest the proposed combination as a valuable approach that would also satisfy the needs of patients. As an alternative, there are also non-invasive stimulation methods that seem to ameliorate tinnitus symptoms (for a recent case study see Richter et al., 2017).

# CONCLUSION

Our interdisciplinary day care tinnitus treatment represents a treatment for patients with chronic tinnitus that reduces tinnitus annoyance. After initial interdisciplinary diagnostic procedures and a first brief tinnitus-specific counseling, a small reduction in tinnitus annoyance was found. A clinically relevant change in tinnitus annoyance was observed between the beginning and the end of treatment and remained stable at least for 6 months.

The best treatment outcome was reached by patients with moderate and severe tinnitus. The improvement in tinnitus annoyance in patients with sick leave within 6 months before treatment onset or with very severe tinnitus annoyance was smaller than for the rest of the investigated population.

Given the high heterogeneity of tinnitus, we predict that the development of adapted JITT to individual needs will be challenging. Additional measurements of neurophysiological correlates might help in understanding which aspects of the symptomatology and the underlying neural network undergo changes in response to treatment and which do not.

# AUTHOR CONTRIBUTIONS

DI: Substantial contributions to the conception and design of the work; the acquisition, analysis and interpretation of data for the work; drafting the work and revising it critically for important intellectual content. Wrote the manuscript. CD: Contribution to the analysis and interpretation of data; drafting the work and revising it critically for important intellectual content. Wrote the manuscript. GV, BM and DR: Substantial contributions to the acquisition and interpretation of data for the work; revising the work critically for important intellectual content. US: Substantial contributions to the conception of the work; revising the work critically for important intellectual content. OG: Substantial contributions to the conception and design of the work; the acquisition, analysis and interpretation of data for the work; revising the work critically for important intellectual content. All authors gave their final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

# ACKNOWLEDGMENTS

The authors would like to thank Marlen Hagemann for the support in acquisition of data and Holger Pickel for organizing the database.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnagi. 2017.00192/full#supplementary-material

# REFERENCES

https://www.biap.org/de/empfehlungen/empfehlungen/ct-02-classificationdes-deficiences-auditives-1/55-02-1-audiometric-classification-of-hearingimpairments/file (Accessed May 15,2017).

**Conflict of Interest Statement:** DI received funding from ISMA GmbH (founder of Terzo hearing therapy) for lecturing their associates about tinnitus. ISMA GmbH had no influence on the presented study.

The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Ivansic, Dobel, Volk, Reinhardt, Müller, Smolenski and Guntinas-Lichius. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparison of the Long-Term Effect of Positioning the Cathode in tDCS in Tinnitus Patients

Sarah Rabau1,2\*, Giriraj S. Shekhawat 3,4,5 , Mohamed Aboseria6 , Daniel Griepp6 , Vincent Van Rompaey 1,2 , Marom Bikson6 and Paul Van de Heyning1,2

1University Department of Otorhinolaryngology and Head and Neck Surgery, Antwerp University Hospital, Edegem, Belgium, 2Faculty of Medicine, Campus Drie Eiken, University of Antwerp, Antwerp, Belgium, 3Section of Audiology/Health Systems, University of Auckland, Auckland, New Zealand, 4Centre for Brain Research, University of Auckland, Auckland, New Zealand, 5Tinnitus Research Initiative, Regensburg, Germany , 6Department of Biomedical Engineering, City College of New York, The City University of New York, New York, NY, United States

Objective: Transcranial direct current stimulation (tDCS) is one of the methods described in the literature to decrease the perceived loudness and distress caused by tinnitus. However, the main effect is not clear and the number of responders to the treatment is variable. The objective of the present study was to investigate the effect of the placement of the cathode on the outcome measurements.

Methods: Patients considered for the trial were chronic non-pulsatile tinnitus patients with complaints for more than 3 months and a Tinnitus Functional Index (TFI) score that exceeded 25. The anode was placed on the right dorsolateral prefrontal cortex (DLPFC). In the first group—"bifrontal"—the cathode was placed on the left DLPFC, while in the second group—"shoulder"—the cathode was placed on the shoulder. Each patient received two sessions of tDCS weekly and eight sessions in total. Evaluations took place on the first visit for an ENT consultation, at the start of therapy, after eight sessions of tDCS and at the follow-up visit, which took place 84 days after the start of the therapy. Subjective outcome measures such as TFI, Visual Analog Scales (VAS) for loudness and percentage of consciousness of tinnitus were administered in every patient.

Results: There was no difference in the results for tinnitus loudness and the distress experienced between the placement of the cathode on the left DLPFC or on the shoulder. In addition, no statistically significant overall effect was found between the four test points. However, up to 39.1% of the patients experienced a decrease in loudness, measured by the VAS for loudness. Moreover, 72% of those in the bifrontal group, but only 46.2% of those in the shoulder group reported some improvement in distress.

Conclusion: While some improvement was noted, this was not statistically significant. Both electrode placements stimulated the right side of the hippocampus, which could be responsible for the effect found in both groups. Further research should rule out the placebo effect and investigate alternative electrode positions.

Keywords: neuromodulation, tDCS, electrode placement

#### Edited by:

P. Hemachandra Reddy, Texas Tech University Health Sciences Center, United States

#### Reviewed by:

Yang-soo Yoon, Texas Tech University Health Sciences Center, United States Samba Reddy, Texas A&M Health Science Center, United States

#### \*Correspondence:

Sarah Rabau<EMAIL_ADDRESS>
Received: 24 October 2016 Accepted: 21 June 2017 Published: 28 July 2017

#### Citation:

Rabau S, Shekhawat GS, Aboseria M, Griepp D, Van Rompaey V, Bikson M and Van de Heyning P (2017) Comparison of the Long-Term Effect of Positioning the Cathode in tDCS in Tinnitus Patients. Front. Aging Neurosci. 9:217. doi: 10.3389/fnagi.2017.00217

# INTRODUCTION

Tinnitus is the perception of sound in the absence of a corresponding external sound source (Eggermont and Roberts, 2004) and is a very common problem. Approximately 25.3% of the US population reports having tinnitus, while 7.9% experience tinnitus frequently (Shargorodsky et al., 2010). Tinnitus affects daily activities and can lead to a high level of distress. The literature describes several mechanisms and structures that could be involved in tinnitus. However, the findings reported cannot always be repeated and the proposed brain structures do not always match. However, it is certain that it is not only the auditory system which is responsible for inducing tinnitus (Jastreboff, 1990). Multiple non-auditory systems involved in cognition, emotion and memory play an important role (De Ridder et al., 2014).

Currently, no treatment is available that eliminates tinnitus completely. Most researchers and clinicians focus on diminishing the level of disturbance experienced and/or the loudness of the tinnitus. One method that can be used is neuromodulation, namely transcranial Direct Current Stimulation (tDCS). In the case of tDCS, the current is applied to the brain by means of two electrodes. The goal of tDCS is to influence those regions involved in tinnitus and consequently lessen the distress caused by, or the loudness of, tinnitus. Previous research has shown that bifrontal tDCS strengthens deficient inhibitory top-down mechanisms in tinnitus and interferes with the emotional processing of tinnitus (Tanaka et al., 2008; Vanneste et al., 2010). However, in the literature tDCS is reported as having a variable effect, with the percentage of responders ranging from 0% to almost 47% (Fregni et al., 2006; Vanneste et al., 2011; Song et al., 2012). The difference in outcomes might be due to different factors such as orientation of the current field, electrode positions, electrode size, stimulation duration and current intensity (Nitsche et al., 2008). Depending on the orientation of the current field, tDCS can either increase or decrease the excitability. Under the cathode, excitability is decreased due to neural hyperpolarization. However, under the anode, excitability is enhanced due to neural depolarization (Nitsche and Paulus, 2000).

The literature also reports the use of different electrode positions to treat tinnitus, with bifrontal tDCS and left temporoparietal area (LTA) tDCS most frequently applied in tinnitus patients. In bifrontal tDCS, the anode and cathode are placed on the right and left dorsolateral prefrontal cortex (DLPFC), respectively. In LTA tDCS, the anode is placed on the left temporal area. Song et al. (2012) found that LTA and bifrontal positioning produced similar results with respect to the percentage of responders and a reduction in tinnitus intensity (Song et al., 2012). Furthermore, the current intensity and duration can also play an important role. Shekhawat et al. (2013) concluded that 2 mA anodal tDCS at LTA for 20 min was the most effective setting for transient tinnitus suppression (Shekhawat et al., 2013).

Concluding, more research is needed towards parameters of tDCS to optimize the effect of it. In that interest, a computational model was calculated to predict differences in the current pathway of two placement montages, namely the ''bifrontal'' montage and the ''shoulder'' montage. The ''bifrontal'' group had the anode and cathode positioned over the F4 (right DLPFC) and F3 (left DLPFC) positions respectively; in the ''shoulder'' group, the anode electrode was placed over F4 and the cathode on the left shoulder. Considering these differences, interest was raised towards the difference in the perception outcome of the tinnitus patients undergoing tDCS using these two different montages. The objective of the present study was to compare the outcomes of the placement of the cathode on the left DLPFC vs. the shoulder.

## MATERIALS AND METHODS

## Subjects

Patients considered for the trial were chronic non-pulsatile tinnitus patients who had complaints for more than 3 months and a Tinnitus Functional Index (TFI) score that exceeded 25. Written informed consent was obtained from every patient (n = 65). The study protocol was approved by the ethical committee of Antwerp University Hospital. Of the 65 subjects, six were lost to follow-up for various reasons. The median age of the subjects was 52 years, within a range of 23 years to 70 years. The median duration of tinnitus was 28 months and fluctuated between 3 months and 264 months. In total 59 subjects—41 men and 18 women were included and randomized based on the parameters of age, TFI score, etiology, gender and degree of hearing loss. In order to create two equal groups, the MS-DOS program MINIM (by S. Evans, P. Royston and S. Day) was used to allocate the subjects by minimization. A flow diagram is presented in **Figure 1**.

## Transcranial Direct Current Stimulation

In the first group—''bifrontal''—the anode and the cathode were placed on the right and left DLPFC, respectively (F4 and F3), while in the second group—''shoulder''—the cathode was placed on the left upper arm, according to the 10–20 international system for EEG electrode placement. Each patient received two sessions of tDCS weekly and a total of eight sessions. The direct current was transferred by means of two saline-soaked pairs of surface sponge electrodes (35 cm3 ) and delivered by a specially developed, battery-driven constant current Neuroconn stimulator (neuroConn, Ilmenau, Germany), with a maximum output of 10 mA. A constant current of 2 mA was applied for 20 min with a fade-in and fade-out of 10 s. **Figure 2** shows the current pattern in both cathode placements. In the bifrontal group, the DLPFC and the hippocampus are stimulated, but not the cingulate cortex. In contrast, in the shoulder placement, the temporal lobe is stimulated, in addition to the cingulate cortex and the right side of the hippocampus.

#### Outcome Measures

Evaluations took place on the first visit for an ENT consultation (Delay), at the start of therapy (Start), after eight sessions of tDCS (End) and 84 days after the start of the therapy (FUV). The delay was added to test spontaneous recovery after visiting an ENTspecialist. The FUV was added to measure the long-term effect of tDCS. The flow diagram is presented in **Figure 1**.

The subjective outcome measures of TFI (Meikle et al., 2012; Rabau et al., 2014), Visual Analog Scales (VAS) for loudness and the Hyperacusis Questionnaire (HQ; Khalfa et al., 2002) were completed for every patient. The Dutch version of the TFI, a self-report questionnaire consisting of 25 questions, was chosen as the primary outcome measurement. A reduction of 13 points on the TFI is considered a meaningful reduction in annoyance experienced by the patient. In addition to a total score, scores of the subscales of intrusiveness, reduced sense of control, cognitive interference, sleep disturbance, auditory difficulties attributed to tinnitus, interference with relaxation, reduced quality of life and emotional distress can also be calculated (Meikle et al., 2012).

The secondary outcome measures were the VAS for loudness and the HQ. On the VAS the patient had to rate the maximum and mean loudness of their tinnitus on a scale of 0–10, with 0 indicating a very soft sound that is not audible and 10 meaning as loud as possible—the tinnitus could not be any louder. The HQ surveys the over-sensitivity to sounds, and consists of 14 items with four answer possibilities: no (0 points); yes, a little (1 point); yes, quite a lot (2 points) and yes, a lot (3 points). The maximum score is 42, with Khalfa et al. (2002) suggesting a cut-off score of 28. Patients scoring more than 28 are considered to have hyperacusis (Khalfa et al., 2002).

#### Statistical Analysis

Statistical analyses were performed on the data to determine whether one placement was preferable to the other. The data were analyzed using SPSS statistical software version 22 MAC OS X (IBM; Armonk, NY, USA). Normal distribution of the data was checked using the Shapiro-Wilk test and Q-Q plots. For each of the outcome measurements, a repeated measures ANOVA was performed with test moment as within subject and group (bifrontal vs. shoulder) as between subject variable. The interaction between test moment and group were added to the model. To assess the impact of the demographic details, covariants (hearing loss and age) were added into the model. The significance level was set at p < 0.05. Descriptive statistics was used to compare the characteristics of the subjects.

hippocampus.

# Computational Model

A finite element montages were generated in COMSOL multi physics 4.2 for analysis based on previous procedures (Truong et al., 2012a,b). Briefly, initially, a 3-D 1 mm∗ 1 mm∗ 1 mm T1 MRI of an adult male was segmented into different head regions using both automated segmentation algorithms and manual segmentation techniques available in the ScanIP software (Simpleware, Ltd., Exeter, UK) to correct for segmentation errors from the automated algorithms, add fat, segment a number of brain deep structures. 5 × 7 cm sponge pads were then placed on F4 and F3 for the ''bifrontal'' montage, and F4 and the left shoulder for the ''shoulder'' montage using ScanCAD (Simpleware, Ltd., Exeter, UK). The mesh was then generated and imported into COMSOL multi physics 4.2 (COMSOL, Inc., Burlington, MA, USA). In COMSOL, the segmented regions were assigned a conductivity (S/m): air, 10−15; skin, 0.465; fat, 0.025; skull, 0.01; CSF, 1.65; gray matter, 0.276; or white matter, 0.126. The electrodes were assigned a conductivity of 5.99 × 107 S/m and the sponges were modeled using the conductivity of saline which is 1.4 S/m. Boundary conditions were set such that the cathode (F3 for ''bifrontal''; left shoulder electrode for ''shoulder'') was the ground and a total of 2 mA of current was applied from the F4 anode. The model was solved and cortical electric field magnitude was plotted for

each montage to analyze the differences between the two montages.

# RESULTS

Repeated measures showed no statistically significant difference between both groups with regard to the TFI, the subscales of the TFI, the VAS for maximum and mean loudness, the percentage of consciousness, or problems experienced falling asleep and waking up during the night (p > 0.05). Looking at the overall effect, no significant effect was found between the test moments for the TFI total score (**Figure 3**), the TFI subscales of intrusiveness, cognitive interference, sleep disturbance, auditory difficulties, quality of life and emotional distress, or with the maximum (**Figure 4**) and mean VAS for loudness and percentage of consciousness (p > 0.05). For the subscale of sleep disturbance (p = 0.048), a statistically significant effect was found between the Start (mean = 64.03) and the FUV (mean = 52.50) test moments in the bifrontal group (**Figure 5**). Concerning the shoulder group, a statistically significant effect was found for the subscale of intrusiveness (p = 0.040) between the Delay (mean = 72.50) and the FUV (mean = 62.50) test moments. The results are shown in **Figure 6**. No statistically significant effect of the demographic details age and hearing loss was found on the outcome measurements (p > 0.05).

recovery or placebo effect found between the Delay (median = 73.30) and Start (median = 63.33) test moments.

Focusing on the measurements before and after the eight sessions of tDCS, we found that 26.8% (n = 7) of the bifrontal group and 37.5% (n = 9) of the shoulder group experienced an improvement of 1 or more on the VAS for maximum loudness, while 30.6% (n = 8) of the subjects in the bifrontal group and 39.1% (n = 9) of the subjects in the shoulder group showed an improvement on the VAS for mean loudness. With respect to the change in TFI scores, 16% (n = 4) of the bifrontal group and 8.4% (n = 2) of the shoulder group showed a clinically significant improvement of more than 13 points, while 56% (n = 14) of the bifrontal group and 37.8% (n = 9) of the shoulder group showed an improvement of less than 13 points on the TFI.

# DISCUSSION

The present study failed to find any difference between the electrode placements with respect to their effects on tinnitus distress and tinnitus loudness. One possible explanation for this may be the limited focus of the tDCS, due to the large electrodes used (Nitsche et al., 2008). The lack of focus prevents the stimulation of a specific area of the brain. Moliadze et al. (2010) have suggested that an extracephalic reference electrode has the potential to better focus stimulation (Moliadze et al., 2010), but direct proof is lacking. In contrast, Parazzini et al. (2012) argued that if the anode and cathode are placed on the far sides of the brain this results in a more widely spread current/electric field (Parazzini et al., 2012). Bikson has studied the role of the position of the return electrode, finding that the repositioning of the return electrode from the contralateral forehead to the upper arm caused the relocation of the current from the frontal regions to the more posterior regions of the brain (Bikson et al., 2010). This finding is consistent with our current pattern, as presented in **Figure 2**. Tissue resistance and altered current flow can also play an important role (Nitsche and Paulus, 2011). Moliadze et al. (2010) claimed that higher current intensity is required to induce identical aftereffects in comparison to bicephalic electrode placement (Moliadze et al., 2010). In relation to bicephalic electrode placement, Shekhawat et al. (2013) concluded that 2 mA anodal tDCS at the LTA for 20 min was sufficient (Shekhawat et al., 2013). Further research should determine whether 2 mA is sufficient to induce aftereffects in the case of extracephalic reference electrode placement and the best dose-response ratio with this electrode position.

No statistically significant overall effect was found between the four test moments. Only for the TFI subscale of sleep disturbance in the bifrontal group was a significant effect found between the Start (median = 70.00) and FUV (median = 51.67) test moments. The statistically significant difference found for the subscale of intrusiveness in the shoulder group cannot be attributed to the tDCS therapy, but to spontaneous recovery or a placebo effect between the Delay (median = 73.30) and Start (median = 63.33) test moments. To rule out a placebo effect, we recommend including a control group in the study protocol for further research. The percentage of responders varied from 26.8% to 39.1% for tinnitus loudness. For the outcome measurement of distress, a clinically significant effect was only found in 16% and 8.4% of the subjects in the bifrontal and shoulder groups respectively. None of these results were statistically significant. The results confirm previous findings reported in the literature, which suggest no long-term effects on distress. However, a positive transient effect of bifrontal tDCS has been reported for tinnitus intensity (Garin et al., 2011; Frank et al., 2012). The present study failed to repeat these results. Most studies have reported a positive effect of tDCS on tinnitus loudness and distress immediately after a tDCS session (Vanneste et al., 2010; Vanneste and De Ridder, 2011; Joos et al., 2014); however, in this study, we focused on the long-term effect.

Another explanation for the lack of statistically significant results may be related to the factors that cause the variance of responders. For tDCS to be considered useful in a clinical setting, it would be interesting to know what causes this variance in the percentage of responders. Further research should also determine what might improve the administration of tDCS to attain clinically significant outcomes. High definition tDCS seems to be a promising alternative in this respect (Shekhawat et al., 2016).

While some improvement was found in both groups, the difference was not statistically significant. This finding would suggest that the area that was stimulated in both groups can be hold responsible for this improvement, namely the right side of the hippocampus, which is the part of the brain that is involved in learning and memory processes. Jastreboff (1990) has pointed out that in addition to the auditory system, the limbic system, including the hippocampus, is responsible for the preseverance of tinnitus. The gating model proposed by Rauschecker et al. (2010) also makes predictions about the structures involved in tinnitus. Although the hippocampus is not a part of the model presented, this structure may be involved in tinnitus but not sufficient to cause tinnitus on its own (Rauschecker et al., 2010; Adjamian et al., 2014). The positioning of an electrode such that it influences the structures involved in the proposed gating model would be an interesting topic for further research. In this study we did not investigate the underlying structures activated during the tDCS stimulation during the two montages (cathode on L DLPFC and shoulder). However, in future it would be insightful to incorporate MRI scans to investigate the neurophysiological structures involved during the tDCS. Another possible explanation is that the effect found can be attributed to a placebo effect. Further research should include a control group to rule out the placebo effect and reveal the real effect of tDCS.

### CONCLUSION

There was no significant difference in outcomes between the electrode placements. Both placements stimulated the right side of the hippocampus, which could be responsible for the effect found in both groups. Further research should rule out a placebo effect, while alternative electrode positions, as well as high definition tDCS, could reveal the effects of stimulating brain structures that are involved in the gating model proposed by Rauschecker et al. (2010).

# AUTHOR CONTRIBUTIONS

SR, VVR and PV designed and set up the study protocol. SR collected and analyzed the data. SR and MB wrote the manuscript. MA and DG designed the model of the distribution of the electric field of tDCS. GSS, VVR, DG, MA and MB reviewed the manuscript.

# FUNDING

The study was supported by a grant from the TOP-BOF project of the University of Antwerp.

#### REFERENCES

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer YSY and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Rabau, Shekhawat, Aboseria, Griepp, Van Rompaey, Bikson and Van de Heyning. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Tinnitus Treatment with Oxytocin: a Pilot study

*Andreia Aparecida Azevedo1 , Ricardo Rodrigues Figueiredo2 \*, Ana Belen Elgoyhen3,4, Berthold Langguth5 , Norma De Oliveira Penido1 and Winfried Schlee5*

*1Universidade Federal de São Paulo, São Paulo, Brazil, 2 Faculdade de Medicina de Valença, Valença, Brazil, 3 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", CONICET, Buenos Aires, Argentina, 4 Facultad de Medicina, Instituto de Farmacología, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina, 5Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany*

*Edited by:*

*Toshihisa Murofushi, Teikyo University, Japan*

#### *Reviewed by:*

*Yiwen Zheng, University of Otago, New Zealand Yasuhiro Chihara, Raffles Japanese Clinic, Japan*

#### *\*Correspondence:*

*Ricardo Rodrigues Figueiredo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuro-Otology, a section of the journal Frontiers in Neurology*

*Received: 09 May 2017 Accepted: 04 September 2017 Published: 21 September 2017*

#### *Citation:*

*Azevedo AA, Figueiredo RR, Elgoyhen AB, Langguth B, Penido NO and Schlee W (2017) Tinnitus Treatment with Oxytocin: A Pilot Study. Front. Neurol. 8:494. doi: 10.3389/fneur.2017.00494*

Keywords: tinnitus, oxytocin, hearing disorders, pharmacotherapy, nasal sprays

# INTRODUCTION

Tinnitus is a phantom auditory sensation that is not generated by an external stimulus. It is a symptom that affects about 25% of American adults, in a frequent basis for around 8% of them (1). According to current trends of thoughts, tinnitus is a central nervous system phenomenon that follows an initial peripheral damage and may be modulated by many concurring neuronal circuits (2). Neuroimaging studies suggest that tinnitus results from the dynamic interaction between auditory and non-auditory pathways, and the result of this interaction, especially when the limbic and autonomous systems are involved, is the trigger of negative emotional associations and appearance of uncomfortable reactions (3). As yet, there is no Food and Drug Administration (FDA)-approved pharmacological treatment for tinnitus. However, various factors structures such as ion channels, neurotransmitters, and receptors have been proposed to be involved in tinnitus pathophysiology (4). Thus, there is no reasonable argument to believe that tinnitus could not be pharmacologically treated (5).

Oxytocin is a neurohormone that may also act as a neurotransmitter, produced by magnocellular neurons in the ventricular nuclei of the hypothalamus (6). Oxytocin's production is stimulated by high estrogen doses and inhibited by high doses of catecholamines (dopamine, epinephrine, and norepinephrine) (7). It is released to the bloodstream by the posterior pituitary. Oxytocin receptors (OTR) are widespread throughout the human body and belong to the family of heterotrimeric G-protein-coupled receptors, which are found in many cell types (8). OTR promote the inhibition of adenilate-cyclase, thus reducing intracellular levels of cyclic-AMP and subsequently opening potassium channels and closing calcium channels, having, thereby, an inhibitory signature (9).

Oxytocin is responsible for important physiological functions, such as uterine contraction, lactation stimulation, sperm transport, and ejaculation (10). In addition, oxytocin plays a complex role in social cognition and behavior. Important aspects of human social interaction such as empathy, trust, and social learning are influenced by oxytocin (11). Among other mechanisms the prosocial effects of oxytocin are mediated by reduction of amygdale activation (12). Recently, it has been shown, that oxytocin also increases the salience of acoustic social stimuli by modulating the inhibitory function in the auditory cortex (13).

Imaging studies have demonstrated that tinnitus loudness and tinnitus distress are reflected by increased activation in networks involving the auditory cortex and the amygdala, respectively (3, 14). Therefore, we hypothesized that oxytocin may influence tinnitus perception and tested in two studies whether its intranasal application reduces tinnitus loudness and distress.

#### MATERIALS AND METHODS

The trial comprised 2 studies, as follows.

#### Study 1

Study Design Study 1 was designed as an open pilot study.

#### Subjects

Fifteen patients who presented with the primary complaint of tinnitus in a local ENT clinic in São Paulo and Valença were included. Only patients older than 18 years with a continuous perception of tinnitus of at least 6 months duration were included. Patients were excluded if their Tinnitus Handicap Inventory (THI) (15) score in the Brazilian Portuguese validated version (16) was below 16 points. All patients underwent a complete otolaryngological evaluation and were excluded from the study in case of external or media otitis or in the presence of A-s, B, and C tympanogram curves. Also, women in fertile age [up to 49 years old (17)] were excluded from the study, to avoid oxytocin-induced uterine contractions. Informed consent was obtained prior to the study, and the patients were informed about the clinical procedures. Clinical and demographic characteristics of participants (mean age, percentage of females, mean duration of tinnitus in months, laterality and scores on the THI in both studies) are described in **Table 1**.

#### Experimental Procedure

Oxytocin (Syntocinon) was administered daily for a duration of 10 weeks. Patients were instructed to apply one puff [each puff corresponds to 4 oxytocin IU] of oxytocin in each nostril two times a day, which sums up to a dosage of 16 IU per day. The patients were asked to store the oxytocin in the refrigerator throughout the study period. Subsequent visits were made in weeks 1, 2, 3, 4, 6, 8, and 10 to monitor study progress and screen for side effects. Treatment effects were assessed with the THI (primary outcome measurement) and the Clinical Global Impression (CGI) scale improvement [secondary outcome measurement; CGI-I (18)]. The CGI scale is a well-accepted instrument in clinical research for evaluating the clinical improvement retrospectively. The instruction in the CGI is: "Please rate the total improvement of your tinnitus complaints compared to before beginning of treatment."

#### Statistical Analysis

For the THI, a mixed model analysis of variance (ANOVA) was calculated with the factor time and a random intercept per patient. A mixed model ANOVA was also calculated for the CGI with the factor time (weeks 1–10) and a random intercept per patient. In case of positive results of the ANOVA further comparisons were calculated.

#### Study 2

During study 1, five subjects reported an immediate effect (5–10 min after drug administration) on tinnitus sensation (around 50% reduction of tinnitus volume). Considering this unexpected finding, we decided to investigate this tinnitus reduction following one single dose in more detail. Therefore, we performed a second study with a single dose of oxytocin treatment in a double-blind placebo-controlled study using a crossover design. As study 1 turned out with positive results, study 2 was performed with a similar number of patients.

*THI, Tinnitus Handicap Inventory.*

#### Subjects

Study 2 was performed in the same ENT clinic as study 1. Seventeen patients with the primary complaint of tinnitus and who had not participated in study 1 were included.

As in the first study, all patients were older than 18 years and reported chronic tinnitus of more than 6 months duration. Prior to the study, all patients were seen by an experienced ENT doctor. Patients with vascular or muscular origin of their tinnitus, patients with conductive hearing loss and patients with regular intake of other medications during the study period were not included. Patients reporting previous experience with oxytocin nasal spray were not included as well. A further inclusion criterion was a tinnitus loudness rating of at least 4, measured on a scale between 0 and 10.

One patient dropped out of the study because she could not return for the second visit. Clinical and demographic characteristics of the 16 patients, which were included in the analysis, are displayed in **Table 1**. Informed consent was obtained prior to study participation.

#### Experimental Procedure

In study 2, the effects of nasal oxytocin administration were compared with placebo treatment. The order of drug treatment was counterbalanced. Patients were randomized to receive either a single dosage administration of 16 IU of oxytocin (Syntocinon) or a placebo treatment with a nasal spray containing distilled water, which is indistinguishable to oxytocin nasal spray considering smell and taste. A randomization table was created prior to study start by Winfried Schlee who is not in contact with the study participants. After an interval of 1 week, the patients received a second administration with either placebo (the group who received oxytocin first) or with oxytocin (the group who received placebo first). The patients, as well as the medical doctor who evaluated the results (author 1), were blinded to the treatment assignment. Author 2, who applied oxytocin and placebo, was not blinded. In the oxytocin condition, patients received two puffs of oxytocin in each nostril, which corresponded to 16 IU. Likewise, in the placebo treatment, the patients received two puffs distilled water in each nostril, which is supposed to have no clinical effect.

The primary outcome measurement was a visual analog scale (VAS) asking for the tinnitus loudness (19). The VAS score for tinnitus loudness was assessed directly before administering the nasal spray as well as 30 min and 24 h after the intervention. Additionally, the CGI-I was applied 30 min and 24 h after the intervention as secondary outcome measurement.

#### Statistical Analysis

For the VAS scores a two-way mixed model ANOVA was calculated with the factors *time* and *treatment*. A random intercept was modeled for each participant. Mixed model ANOVA were also used to analyze the results of the CGI scale. For the CGI scores, again a two-way mixed model ANOVA was calculated with the factors *time* and *treatment*.

The statistical analysis was performed with the statistical software package R, version 3.3.3 [R Core Team (2017). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/]. For the mixed models analysis, the "nlme" library (version 3.1-131) was used. A *p*-value ≤ 0.05 was considered as significant. Correction for multiple comparisons was applied using the stepwise Holm–Bonferroni method (20).

#### Study Registration

The study was approved by the ethics committee and registered at http://clinicaltrials.gov.

#### RESULTS

#### Study 1

Mean scores for the THI and the CGI-I are presented in **Figures 1** and **2**, respectively. Both measures show continuous and significant decreases of tinnitus-related symptoms measured with the THI [*F*(7,98) = 7.45, *p* < 0.0001] as well as global clinical improvement (CGI-I) [*F*(6,84) = 5.22, *p* < 0.0001] over the study period.

For the THI scores, further comparisons using paired *t*-tests between baseline and all other time points reveal significant differences for the THI measurements at week 1, 2, 3, 4, 6, 8, and 10 (*p*-values of all comparisons survived the Holm–Bonferroni method for multiple comparisons).

For the CGI-I additional tests were calculated to test if scores were different from the value of 4, which indicates "no change." No significant difference was found for week 1, week 2, and week 3. The comparisons for week 4, 6, 8, and 10; however, survived the Holm–Bonferroni correction for multiple comparisons. For week 4 and 6, the CGI scores indicated a change with high significance (*p* < 0.01), for week 8 and 10, the CGI scores indicated a change with very high significance (*p* < 0.001).

Paired *t*-tests were calculated to compare the maximum hearing loss of the participants before and after the treatment. The hearing function did not change significantly, neither for the right ear (*p* = 0.2) nor for the left ear (*p* = 0.36).

#### Study 2

Mean scores of the oxytocin treatment and the placebo intervention are presented in **Figures 3** and **4** for the VAS scores and the CGI-I, respectively.

In the ANOVA for the VAS scores, the main effect of *treatment* was highly significant [*F*(1,77) = 9.54, *p* = 0.003] and the main effect of *time* was significant [*F*(2,77) = 4.47, *p* = 0.015]. The interaction effect *time* × *treatment*, however, failed to reach the level of significance [*F*(2,77) = 1.13, *p* = 0.33]. Cohen's *d* effect sizes for the VAS ratings were 0.55 (30 min) and 0.56 (24 h) for oxytocin treatment and 0.18 (30 min) and 0.19 (24 h) for placebo treatment, respectively.

Furthermore, the ANOVA using the CGI scores as dependent variable, the main effect *treatment* was highly significant [*F*(1,46) = 8.72, *p*= 0.005], while the main effect *time* did not reach statistical significance [*F*(1,46) = 0.60, *p* = 0.44]. There was no statistically significant interaction effect [*F*(1,46) = 0.001, *p* > 0.9]. Additional *t*-tests were calculated to confirm the results and showed that there was no statistical significant difference between the two time points (30 m and 24 h after treatment) for the oxytocin (*p* > 0.63) nor for the placebo condition (*p* > 0.58). A *t*-test testing

Figure 1 | Average scores of the Tinnitus Handicap Inventory (THI) (0–100) before the treatment and at all visits during the study (weeks 1–8). The final assessment was done at the end of treatment in week 10.

for the difference between the oxytocin and placebo intervention, irrespective of the time point, revealed a statistically significant improvement for the oxytocin treatment (*t* = −2.9, *p* = 0.005).

#### DISCUSSION

The present results show that in an exploratory non-controlled study, a 10-week treatment with daily intranasal puffs of oxytocin produced a significant reduction in the THI and CGI in a group of tinnitus patients. Moreover, a double-blind controlled study with a single dose of an intranasal oxytocin, although not significant, exhibited a tendency in a reduction in tinnitus distress, as measured by the CGI.

The presented pilot studies were designed to explore possible effects of oxytocin on tinnitus. The open 10 week treatment study (study 1) suggests that regular intake of oxytocin can reduce tinnitus-related handicap. Open pilot studies have been suggested as a useful screening tool to identify potentially promising pharmacological compounds (21–23). Motivated by the promising results of study 1, we performed also a placebo-controlled crossover trial to investigate the short-term effects of a single dose of oxytocin. Whereas questionnaires represent the gold standard for assessing longer lasting effects of therapeutic interventions (24), short-term effects can be best detected with VAS. While the clinical effects of the short-term oxytocin treatment are much smaller than the long-term effects in study 1, they still suggest after).

a potential therapeutic role of oxytocin for tinnitus. These preliminary results warrant further controlled studies, which should include more patients and investigate chronic treatments with oxytocin. The results of the presented pilot studies provide an estimation of the effect sizes for both acute and long-term effects of oxytocin to inform the study design of future randomized controlled studies. While the open study with the long-term treatment (study 1) revealed a strong reduction of tinnitusrelated distress over time, the short-term treatment (study 2) revealed only small differences between placebo and oxytocin. Further studies on oxytocin treatment for tinnitus should invest in long-term treatment.

A wide variety of compounds are used off-label to treat tinnitus patients (5, 25). However, there is still no US FDA- or European Medicines Agency-approved drug on the market for this clinical unmet use (5). The comprehensive list of compounds includes almost the entire pharmacopeia arsenal, such as anxiolytics, anticonvulsants, antidepressants, *N*-methyl d-aspartate (NMDA) antagonists, cholinergic antagonists, antihistamines, vasodilators, antipsychotics, sodium and calcium channel antagonists, antidiuretics, and herbal medicines, among others (5, 25). In most cases, the pharmacological treatment is used to treat comorbidities which accompany tinnitus, such as frustration, annoyance, anxiety, depression, irritation, concentration difficulties, and sleep disturbances which are most relevant for the perceived tinnitus severity (5, 19). In this context, the exploratory use of oxytocin on tinnitus patients is well justified, since it is a compound with a different mechanism of action from those previously tested.

In particular, Kirsch et al. have shown that when compared with placebo, oxytocin potently reduces activation of the amygdala and coupling of the amygdala to brainstem regions implicated in autonomic and behavioral manifestations of fear (26). In this regard, tinnitus neuroimaging techniques have identified brain networks related to tinnitus associated distress which include the amygdala and the autonomic nervous system (3). Recently, it has been shown that tinnitus distress correlates with enhanced effective connectivity from the amygdala to the auditory cortex (27). An EEG analysis revealed that tinnitus distress is correlated with more synchronized alpha activity in various emotion-related areas, including the subcallosal anterior cingulate cortex, the insula, the parahippocampal area and the amygdale (28). In addition, an MEG functional connectivity analysis has shown long-range coupling between frontal, parietal and cingulate brain areas in alpha and gamma phase synchronization related to tinnitus distress (29). In summary, tinnitus distress has been related to the co-activation of a network which includes amygdala, anterior cingulate cortex, insula, parahippocampal area and which is under direct influence of the posterior cingulate and prefrontal cortex (30, 31). This network partially overlaps with brain areas implicated in distress in patients suffering from pain (32), dyspnea in asthma (33), somatoform disorders (34) and might, therefore, represent a non-specific distress network.

Through source localized resting-state EEG and electrocardiogram recordings, the participation of the autonomic nervous system to tinnitus distress has been investigated, showing that the dorsal and subgenual anterior cingulate, as well as the left and right insula are important in the central control of the autonomic system in tinnitus patients (35, 36). The perceived distress in tinnitus patients seems to be sympathetically mediated. It is of interest that the same areas that are involved in the control of the autonomic system are also involved in salience processing and distress. This suggests that the autonomic nervous system critically influences salience perception and distress associated with tinnitus.

Thus, the observed reduction of tinnitus handicap after longterm oxytocin application could be explained by a modulatory influence of oxytocin on amygdala activity and on a decoupling between amygdala activation and autonomous nervous system regulation. Further studies investigating the effect of oxytocin on perception and simultaneously on brain activity and connectivity could shed further light on the mechanisms by which oxytocin interacts with tinnitus perception and distress.

#### ETHICS STATEMENT

Comitê de Ética em Pesquisa Médica do Centro Universitário de Volta Redonda—Unifoa/Fundação Parecer No. 42257214.1.0000. 5237/1.017.546 7/4/2015.

#### REFERENCES

## AUTHOR CONTRIBUTIONS

Study design: AA, RF, WS, BL, and AE. Data collection: AA and RF. Statistics: WS. Writing: AA, RF, AE, BL, and NP. Manuscript revision: AA, RF, AE, BL, and PS. All contributed for writing and revision: AA, RF, BL, AE, NP, and WS.

# FUNDING

This work was supported by a grant from Tinnitus Research Initiative.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Azevedo, Figueiredo, Elgoyhen, Langguth, Penido and Schlee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Identification of Candidate Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor Which May Improve Vagus Nerve Stimulation in Chronic Tinnitus

Tijana Bojic´ 1 \*, Vladimir R. Perovic´ 2 , Milan Sencanski ´ 2 and Sanja Glišic´ 2

1 Laboratory of Radiobiology and Molecular Genetics, Institute of Nuclear Sciences Vinca, University of Belgrade, Belgrade, ˇ Serbia, 2 Center for Multidisciplinary Research, Institute of Nuclear Sciences Vinca, University of Belgrade, Belgrade, Serbia ˇ

#### Edited by:

Winfried Schlee, University of Regensburg, Germany

#### Reviewed by:

Andrea Harrington, University of Adelaide, Australia Ricardo Rodrigues Figueiredo, Faculdade de Medicina de Valença, Brazil

> \*Correspondence: Tijana Bojic´<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neuroscience

Received: 22 June 2017 Accepted: 02 November 2017 Published: 14 November 2017

#### Citation:

Bojic T, Perovi ´ c VR, Sen ´ canski M and ´ Glišic S (2017) Identification of ´ Candidate Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor Which May Improve Vagus Nerve Stimulation in Chronic Tinnitus. Front. Neurosci. 11:636. doi: 10.3389/fnins.2017.00636 Chronic tinnitus is characterized by neuroplastic changes of the auditory cortex. A promising method for therapy of chronic tinnitus is vagus nerve stimulation (VNS) combined with auditory stimulation. The principle of VNS is reversal of pathological neuroplastic changes of the auditory cortex toward physiological neural activity and synchronicity. The VNS mechanism of action in chronic tinnitus patients is prevailingly through the muscarinic neuromodulation of the auditory cortex by the activation of nc. basalis Meynerti. The aim of this study is to propose potential pharmaceutics which may improve the neuromodulatory effects of VNS. The working hypothesis is that M1 receptors have a dominant role in the neural plasticity of the auditory cortex. We propose that allosteric agonists of the muscarinic receptor type 1 (M1) receptor could improve specificity and selectivity of the neuromodulatory effect of VNS on the auditory cortex of chronic tinnitus patients even in the circumstances of lower acetylcholine brain concentration. This intervention would also reinforce the re-learning process of tinnitus (sub)networks by acting on cholinergic memory and learning mechanisms. We performed in silico screening of drug space using the EIIP/AQVN filter and selected 50 drugs as candidates for allosteric modulators of muscarinic receptors. Further filtering of these compounds by means of 3D QSAR and docking revealed 3 approved drugs—bromazepam, estazolam and flumazenil as the most promising candidates for combined chronic tinnitus therapy. These drugs should be further evaluated by biological tests and clinical trials.

Keywords: tinnitus, muscarinic allosteric agonists, M1 receptor, vagus nerve stimulation, in silico analysis, information spectrum method

# INTRODUCTION

Tinnitus, the perception of phantom sound, is a debilitating condition with notable prevalence: approximately 10–15% of the general population experience tinnitus and for 7 million this is a debilitating condition (Geven et al., 2014). The Royal National Institute for Deaf People estimates that 13 million people in Western Europe and USA seek medical assistance for their tinnitus symptoms (Vio and Holme, 2005). Demographic development and increase of occupational and ambient noise support the view that this problem will be even more prevalent in the future. In spite of the fact that more than 4 million prescriptions are written for the treatment of tinnitus, there is not a single drug approved by the FDA specific for the treatment of tinnitus. The estimates are that a potential tinnitus drug could have a product value of more than \$600 million in the first year after its release (https:// www.ata.org/understanding-facts).

The problem of chronic tinnitus is mainly of central origin. The pathophysiological substrate of chronic tinnitus is still a matter of intense debate (De Ridder et al., 2014b; Norena, 2015). The majority of chronic states of tinnitus begin with the functional damage of the cochlea (i.e., noise or hearing loss) and consequent abnormal input to the higher level neural structures of the auditory pathway (Eggermont and Roberts, 2004; Guitton, 2012; Chen et al., 2013; Wu et al., 2016). When tinnitus stabilizes as a chronic condition (not less than 1 year, Malinvaud et al., 2016), then numerous functional disarrangements occur, like the hyperpolarization of thalamic relay cells (Llinas et al., 2005), the changes in the central neural sensitivity or gain (Norena, 2011) and the functional coupling of different parallel brain networks (Kalauzi et al., 2012, 2014) into the "tinnitus (sub)network" (De Ridder et al., 2014b). This finally results also with the functional and anatomical disarrangement of the auditory cortex tonotopic map (Norena and Eggermont, 2005; Guitton, 2012).

The central (Zoccoli et al., 2005; Bojic et al., 2016 ´ ) and peripheral components of the autonomic nervous system (Bojic, ´ 2003; Silvani et al., 2003; Platiša et al., 2016) play crucial roles in the stabilization and manifestations of chronic tinnitus (Jastreboff, 2011). Cholinergic innervation plays a major role in the development and plasticity of the auditory cortex (Shideler and Yan, 2010). The growth and functional coupling of cholinergic innervations with auditory cortical cells goes in parallel with the forming of thalamocortical connections during embryological development and in the early stage after the birth. The presence of muscarinic acetylcholine receptors plays a crucial role in this process: muscarinic receptor type 1 (M1) regulates the expression of different neurotrophins (brainderived neurotrophic factor and nerve growth factor; Da Penha Berzaghi et al., 1993; Betancourt et al., 2006), determines the structure of neurons by promoting cell survival (Tobin and Budd, 2003) and stimulates the neural (VanDeMark et al., 2009) and dendritic outgrowth (Zhang et al., 2005). Finally, muscarinic antagonists decrease the frequency specific plasticity of the auditory cortex in the paradigm of auditory fear conditioning (Ji et al., 2005; Ji and Suga, 2009) and, importantly for this concept, the stimulation of nc. basalis Meynerti (Bakin and Weinberger, 1996; Weinberger, 2003). Nc. basalis Meynerti electrical stimulation paired with tones acutely enhances cortical neural plasticity and reverses the neurological and perceptual correlates of tinnitus in adult animals (Nichols et al., 2011). M1 impacts also the processes of learning and memory (Zhang et al., 2006; Butcher et al., 2016) which are crucially important for the genesis and maintenance of tinnitus (De Ridder et al., 2014b; Eggermont and Kral, 2016; Vanneste and De Ridder, 2016).

Different techniques, both noninvasive and invasive, were tested in order to reverse the pathological neuroplastic changes of the auditory cortex toward the physiological state (Vanneste and De Ridder, 2012). Vagus nerve stimulation paired with tone stimulation is among the most promising tools for treatment of chronic tinnitus (Engineer et al., 2011; Shetake et al., 2012; De Ridder et al., 2014a). It is based on the principle of provoking the central neuromodulatory responses by stimulation of vagal afferent fibers and nc. basalis Meynerti which abundantly innervate the auditory cortex. Vagus stimulation paired with tone stimulation has an effect of targeted plasticity, the phenomenon of reversing the map changes in individuals with tinnitus. The area of tinnitus specific cortical neurons is consequently diminished (Engineer et al., 2011). In human studies the efficiency of this method to reduce tinnitus severity was around 40%, and interestingly, all the patients who did not experience an improvement were on drug therapy that included, among others, muscarinic antagonists (De Ridder et al., 2014a). More, it is well-documented in the literature that M1 receptors have a crucial role in the experience-dependent plasticity of the auditory cortex (Shideler and Yan, 2010). Our hypothesis is that in silico identification of M1 receptor allosteric agonists would propose a new line for the clinical research for modulated vagus nerve stimulation (VNS) paired with tones, potentially more targeted and more efficient. In addition, by agonizing the cholinergic mechanisms of learning and memory, attention, stress response, wakefulness and sleep and sensory information processing (Ferreira-Vieira et al., 2016), M1 allosteric agonists would stabilize the process of re-learning of neural networks involved in tinnitus perception. In order to choose the best candidates for these drugs we applied in silico strategies that resulted with three candidates for M1 allosteric modulators.

### METHODS

In this paper, we implemented a virtual screening protocol that includes both short and long-range interactions between interacting molecules. The long-range interactions are denoted by the parameters—the average quasi valence number (AQVN) and the electron-ion interaction potential (EIIP).

First, the EIIP/AQVN filter was applied for in silico screening of the DrugBank (http://www.drugbank.ca) (Wishart et al., 2006) and then followed by 3D QSAR and molecular docking for identification of candidate allosteric modulators of M1.

#### EIIP/AQVN

The EIIP for organic molecules can be determined by the following simple equation derived from the "general model pseudopotential" (Veljkovic et al., 2011)

$$\text{EIIP} = 0.25 \, Z^\* \sin(1.04 \, \pi \, Z^\*)/2\pi \tag{1}$$

where Z∗ is the average quasi valence number (AQVN) determined by

$$Z^\* = \sum^{\text{m}} \text{(niZi/N)}\tag{2}$$

where Zi is the valence number of the ith atomic component, ni is the number of atoms of the ith component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values calculated according to Equations (1, 2) are expressed in Rydberg units (Ry).

Further filtering of these compounds was performed by means of 3D QSAR(quantitative structure–activity relationships) in Pentacle software and followed by docking.

#### 3D QSAR

In order to build a pharmacophoric model and select compounds based on their pharmacophoric similarity, 12 literature M1 modulators (Target ID CHEMBL216) were downloaded from the ChEMBL database. All compounds were converted into the SDF format and then imported, along with candidate compounds into the Pentacle QSAR software, protonated at pH 7.4, and oriented according to the principal moments of inertia. Standard GRIND descriptors were calculated and the PCA model was built. From PCA scores that included the first two major components, PC1 and PC2, the most similar drug molecules to literature allosteric modulators were selected for further filtering.

#### Molecular Docking

Molecular docking of selected candidates was performed into the built homology model of the M1 receptor. The binding site of allosteric modulators was identified from the 4MQT structure and was placed in between extracellular loops of the 2 and 3 region. The grid box for docking, with dimensions 20 × 20 × 20 Å was placed to occupy this space. The receptor and ligands were prepared in Autodock Tools 1.5.6. Docking was carried out in AutodockVina (Trott and Olson, 2010). Exhaustiveness was set to 250.

# RESULTS

# EIIP/AQVN Filter

The virtual screening (VS) protocol in this study was based on the application of consecutive filters to select candidate allosteric modulators of M1. Previously it was shown for molecular targets in diverse pathological states that small molecules with similar AQVN and EIIP values interact with the common therapeutic agent (Veljkovic et al., 2011, 2013). This resulted in determining criteria for virtual screening of molecular libraries for compounds with similar therapeutic properties (Veljkovic et al., 2013). The selected learning set consisted of allosteric agonists and positive allosteric modulators of M1, reported in literature (Conn et al., 2009). The compounds from the learning set were inside the active domain with AQVN and EIIP values within the intervals of (3–3.08) and (0.004–0.073), respectively, and this domain was selected as a criterion for the selection of compounds representing candidate allosteric modulators of M1 (**Figure 1**). By applying the EIIP/AQVN-based virtual screening criterion, 50 drugs were chosen out of 1463 approved drugs from the DrugBank (http://www.drugbank.ca) (Wishart et al., 2006).

### QSAR Selection

The 103 molecules after EIIP filtering were subjected to pharmacophoring modeling and selection. Twelve literature M1 modulators were converted into 3D structures, along with drug candidates and GRIND descriptors for all compounds were calculated, from which a PCA model was built (**Table 1**).

From PCA scores that included the first two major components, PC1 and PC2, based on their vicinity in the PC1-PC2 space to literature M1 modulators, the most pharmacologically similar drug molecules were selected. The QSAR filtering was carried in Pentacle software (Duran et al., 2009). Finally, 10 compounds were selected for molecular docking (**Table 2**).

# M1 Muscarinic Receptor Homology Modeling

In order to obtain the relevant structure of the M1 receptor active state, the crystal structure of the M2 muscarinic receptor in the active state with the agonist and allosteric modulator (PDB ID 4MQT) was used as a template for homology modeling. The P11229 sequence of the Homo sapiens muscarinic acetylcholine receptor M1 was used. The modeling was carried out on the Protein Homology/analogY Recognition Engine V 2.0 (Phyre) server (Kelley et al., 2015).

#### Molecular Docking

Ten selected compounds after 3D QSAR were further subjected to molecular docking into the M1 receptor model. Three compounds with the lowest binding energy values (**Table 2**) and identified hydrophilic and hydrophobic interactions with allosteric binding site amino acid residues (Tyr 82, Tyr 85, Tyr 106, Tyr 179, Trp 400, Glu 401) were selected to be the best candidates, along with considerations of drug side effects and purpose. Finally, BROMAZEPAM, ESTAZOLAM,

SSX, Percentage of the X sum of squares; SSXacc, accumulative percentage of the X sum of squares; VarX, percentage of the X variance; VarXaac, accumulative percentage of the X variance.

TABLE 2 | QSAR selection of 10 compounds for molecular docking, with docking energies.

and FLUMAZENIL were found to be the best promising candidates (**Figures 2**–**4**, respectively).

# DISCUSSION

We hypothesized that allosteric activation of M1 receptors in the circumstances of increased cholinergic neurotransmission to the auditory cortex by targeted VNS, will provoke an increase of M1 mediated effects:

The AQVN/EIIP approach was applied previously for the selection of new candidates in pharmacotherapy of the vasovagal syncope (VVS) revealing that the majority of antimuscarinic drugs might have a therapeutical potential for VVS. In this

acetylcholine receptor, with marked aminoacids, and intermolecular interactions. Green: hydrogen bonds, purple: hydrophobic interactions.

study sequential virtual screening criteria were applied, first the AQVN/EIIP based filter for the selection of candidate allosteric agonists of the M1 receptor selecting 50 drugs out of 1463 approved drugs from the DrugBank (http://www.drugbank.ca; Wishart et al., 2006). This step was followed by 3DQSAR analysis and finally docking. The screening identified bromazepam, estazolam, and flumazenil as the most promising drugs which could be repurposed as allosteric agonists of the M1 receptor.

Bromazepam and estazolam are known anti-anxiety agents with a hypnotic effect. Flumazenil has specific antibenzodiazepine action and is used as an antihypnotic agent in circumstances where the benzodiazepine and nonbenzodizepine hypnotic effect has to be reduced.

If we expand the concept to other methods of treatment of tinnitus (invasive or noninvasive; Vanneste and De Ridder, 2012), it is reasonable to propose that pharmacological facilitation of neuromodulatory changes by the in silico identified drugs in our investigation in tinnitus network structures would be beneficial even in these treatments. Future clinical studies of the combined M1 allosteric agonist therapy with VNS and potentially other invasive and noninvasive methods of chronic tinnitus treatment will reveal the final answer regarding their synergistic action.

Learning during wakefulness induces neural plasticity changes. Their stabilization occurs during sleep (Tononi and Cirelli, 2005). The process of sleep selects the synaptic weights of the neural synapses on the basis of their engagement during wakefulness: if the synapses are more used, the sleep process will make them more stabile while the less used synapses during wakefulness will not be strengthened. This is at least partially due to the intensity of cholinergic neurotransmission that

#### REFERENCES

Bakin, J. S., and Weinberger, N. M. (1996). Induction of a physiological memory in the cerebral cortex by stimulation of the nucleus basalis. Proc. Natl. Acad. Sci. U.S.A. 93, 11219–11224. doi: 10.1073/pnas.93.20.11219

increases from wakefulness toward NREM and REM sleep (Hobson and Friston, 2012). The last phase, REM, also known as a dream state, is the state of consciousness where tinnitus is not perceived (De Ridder et al., 2014b). The sleep process in this way supports the mechanisms of learning and memory acquired during wakefulness. If we presume that VNS paired with tones is also the process of re-learning of the cortical networks pathologically changed in tinnitus, then the quality of sleep in chronic tinnitus patients is essential for stabilization of daily acquired neuroplastic changes by the VNS therapy. In line with that, the cholinergic effect together with the hypnotic effect of the proposed drugs could be beneficial and their application justified before bedtime. The caveat that has to be taken into consideration is the feature of benzodiazepines to promote or to reinforce already existing sleep apnea by increased miorelaxation. This effect could diminish the amount of both NREM and REM sleep and potentially prevent the beneficial effect of the sleep on the process of the recovery in tinnitus patients. Biological experiments will address the question of concentration applied to the treatment of tinnitus and the question if sleep apnea as the contraindication for application of the proposed M1 allosteric agonists.

In conclusion, the results presented here suggest a potential novel approach in treatment of chronic tinnitus by VNS paired with tones with in silico repurposed drugs as therapeutic candidates that may target M1 and prospectively improve the treatment.

#### AUTHOR CONTRIBUTIONS

TB, VP, MS, and SG contributed equally to the conception of the work, acquisition, analysis, and interpretation of data. TB, VP, and SG participated in drafting the manuscript, revisiting it critically and gave final approval of the version to be published. The authors reached the agreement to be accountable for all the aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

#### ACKNOWLEDGMENTS

This work was financed by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project III 41028 and 173001. The preliminary results of this article presented at 10th International Tinnitus Research Initiative Conference and 1st EU COST action (TINNET) Conference. Tinnitus: subtypes, mechanisms and interventions, 16–18th March 2016, Nottingham, UK were awarded by MRC Institute of Hearing Research Early Career Award.

Betancourt, A. M., Burgess, S. C., and Carr, R. L. (2006). Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain. Toxicol. Sci. 92, 500–506. doi: 10.1093/toxsci/ kfl004

eds P. L. Parmeggiani and R. Velluti (London: Imperial College Press), 351–369.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Boji´c, Perovi´c, Sen´canski and Gliši´c. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Positive association between Tinnitus and arterial hypertension

*Ricardo Rodrigues Figueiredo1,2\*, Andréia Aparecida Azevedo1 and Norma De Oliveira Penido1*

*1Otolaryngology, Universidade Federal de São Paulo, São Paulo, Brazil, 2Otolaryngology, Faculdade de Medicina de Valença, Valença, Brazil*

#### *Edited by:*

*Winfried Schlee, University of Regensburg, Germany*

#### *Reviewed by:*

*Jae-Jin Song, Seoul National University Bundang Hospital, South Korea Marzena Mielczarek, Medical University of Lodz, Poland*

> *\*Correspondence: Ricardo Rodrigues Figueiredo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuro-otology, a section of the journal Frontiers in Neurology*

*Received: 23 June 2016 Accepted: 21 September 2016 Published: 05 October 2016*

#### *Citation:*

*Figueiredo RR, Azevedo AA and Penido NO (2016) Positive Association between Tinnitus and Arterial Hypertension. Front. Neurol. 7:171. doi: 10.3389/fneur.2016.00171*

Keywords: tinnitus, arterial hypertension, hearing loss, cardiovascular diseases, hearing disorders

# INTRODUCTION

Tinnitus is the perception of noise, which is not generated by external stimulus (1). It affects approximately 25% of the general population: one third on a frequent basis (2). Tinnitus may be classified as auditory and para-auditory tinnitus with the former representing the majority of cases, and the latter being subdivided into muscular and vascular tinnitus, sometimes referred as somatosounds (3).

According to the most recent trends of thought, tinnitus is considered a symptom which may have multiple causes, sometimes even in a single patient (4, 5). Noise exposure, metabolic and cardiovascular disease, presbycusis, ototoxicity, and cranial and cervical trauma are the most frequently considered causes of tinnitus (5, 6). Caffeine abuse, dietary factors, temporomandibular joint, and cervical disease have also been described as contributing factors (7–9).

Systemic arterial hypertension is a multifactorial clinical condition characterized by raised and sustained arterial pressure levels (10). It is defined as systolic levels equal or greater than 140 mmHg and diastolic levels equal or greater than 90 mmHg (10). The prevalence in Brazil, similar to that in other countries, is estimated to be between 22.3 and 43.9% (32.5% average), raising to 50% between 60 and 69 years old and 75% for 70 years and older (10–12). The presence of comorbidities, such as diabetes mellitus and dyslipidemias, and habits, such as smoking, was demonstrated to increase complications risks (13). Arterial hypertension has been described as a possible cause of tinnitus since 1940s (14). Three principle mechanisms suspected of being involved are: damage to inner ear microcirculation (15), ototoxicity by antihypertension drugs (16), and perception of noise generated by blood vessels (3).

As related to inner ear microcirculation, the *stria vascularis* was demonstrated to be the main cochlear site damaged by arterial hypertension (17). Sodium retention could also lead to an increase of extracellular fluid volume, including the perilymph (18), and the endocochlear potential being reduced in hypertensive rats (19). Moreover, hypertension has been associated with a higher risk of hearing loss in brain ischemia (15) and also with a slower recovery in sudden hearing loss (20).

In considering ototoxicity, an extensive review cited diuretics, beta-blockers, angiotensin-conversing enzyme (ACE) inhibitors, angiotensin II receptors blockers, and calcium channels blockers as possible ototoxic medications (21). Furosemide's ototoxicity is the most studied form, producing a quick and reversible decrease of the endocochlear potential (22).

As for vascular tinnitus, some studies cite hypertension as a causal factor, mainly when vascular abnormalities have been ruled out (3). An anatomopathological study demonstrated a high incidence of bony dehiscence of the carotid canal in the middle ear, which may affect the inner ear microcirculation and also generate vascular noises (23).

According to a systematic review, there is evidence of an association between tinnitus and arterial hypertension, but there is a lack of more comprehensive studies (24). The association is stronger in studies that analyzed the presence of arterial hypertension in patients with tinnitus than in those which analyzed the presence of tinnitus in patients with arterial hypertension.

The main purpose of this study is to analyze the presence of arterial hypertension in tinnitus and non-tinnitus patients. Secondary purposes are to analyze differences between tinnitus impact and psychoacoustic measurements in hypertensive and normotensive patients and to evaluate the association between the presence of tinnitus and the diverse antihypertensive drugs employed.

#### MATERIALS AND METHODS

This is a transversal case–control study in which individuals of 18 years of age or older with and without tinnitus were selected at the author's ENT clinic from 2011 to 2014. The trial was approved by the Institutional Review Board (number 010/ CEP-FMV/2011). This study was carried out in accordance with the recommendations of the aforementioned Institutional Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Two groups were created: the first included patients with tinnitus of at least 3 months duration and the second included patients without tinnitus (control). The control group was paired with the tinnitus group for gender, age, and race. The time of tinnitus onset as related to arterial hypertension onset was not an exclusion criteria. Patients from both groups were submitted to anamnesis (including demographics, comorbidities, and habits), otorhinolaryngological physical examination, and arterial pressure measurements with a calibrated sphygmomanometer (Erka Perfekt Aneroid, Germany), in order to exclude possible undiagnosed arterial hypertension. The criteria for blood pressure evaluation were those from the VII Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure, U. S. Department of Health and Human Services, as previously described.

Patients allegedly normotensive with high blood pressure detected at the physical examination were excluded. Patients from both groups also underwent conventional pure tone and speech audiometry.

Tinnitus patients were questioned regarding their tinnitus characteristics (duration, type of sound, laterality, and periodicity) and also classified their tinnitus according to a Visual Analog Scale (VAS), from 1 to 10 (for intensity and distress) and to the Brazilian Portuguese validated version of the Tinnitus Handicap Inventory (THI) (25). They also underwent psychoacoustic measurements of their tinnitus – Pitch Matching (PM) and Minimum Masking Level (MML).

The sample size was determined after the analysis of the arterial hypertension prevalence in a preliminary sample of tinnitus patients (*n* = 46) that was compared to the prevalence of arterial hypertension in the Brazilian general population, obtained from previous studies (10). The minimum number of individuals for each group was determined to be 140.

For whole sample analysis, the comparison of the variables between the tinnitus group and the control group was performed using the Mann–Whitney test for the numerical data and the chisquare or the Fisher test for the categorical data. The Spearman coefficient was performed to analyze the association between the duration of arterial hypertension and the numerical audiometry measurements. The Cochran–Mantel–Haenszel was used to analyze the association between tinnitus and arterial hypertension adjusting for the presence of hearing loss. The significance level was established at 5%, and the statistical analysis was performed with the SAS statistical software, 6.11 version (SAS Institute, Inc., Cary, NC, USA).

#### RESULTS

The final sample was composed of 144 patients in the tinnitus group and 140 in the control group. The tinnitus group was then divided into two subgroups: one for patients with arterial hypertension and the other without arterial hypertension.

The average age was 57.8 years of age for the tinnitus group and 58.6 for the control group. The averages of tinnitus duration, VAS for intensity, VAS for distress, and THI scores for the tinnitus group were, respectively, 54.5 months–4.5 years, 5.45, 5.83, and 41.7 points.

Although the duration of arterial hypertension was longer (average of 128.7 months–10.7 years) than the duration of tinnitus (average of 54.5 months), no statistically significant correlation was found, according to the Spearman coefficient. Considering the patients that could ascertain the duration of both arterial hypertension and tinnitus (*n* = 56), for 42 of them (75%) arterial hypertension preceded tinnitus.

**Table 1** shows the comparison of numerical variables and **Table 2** shows the categorical variables, the latter confirming the pairing by gender, age, and race. As shown in **Table 2**, arterial hypertension was more prevalent in tinnitus patients (*p* = 0.024), demonstrating an association of tinnitus and arterial hypertension. This association was not found when data were adjusted for the presence of hearing loss (*p* = 0.27, according to the Cochran– Mantel–Haenszel test). Hearing loss was more frequent in the tinnitus group, as shown in **Table 3**, but there was no difference between the two groups as to the type of hearing loss or the shape of the audiogram curve. Sensorineural descendant was the most frequent curve in both groups (75% in the tinnitus patients group and 58% in the control group). Also, there was no difference in the speech recognition index of both groups (100% median in both).

Table 1 | Analysis of the numerical variables (age, duration of arterial hypertension, and daily consumption of caffeine) according to the group.

*Significant values in bold.*

*IQA, Interquartilic Amplitude: Q1–Q3.*

Table 2 | Analysis of categorical variables (gender, age, race, and presence of arterial hypertension) according to the groups.

*Significant values in bold.*

*a* χ*2 or Fisher test.*

The analysis of the antihypertensive drugs used in both groups is shown in **Table 4**.

The analysis of comorbidities and habits demonstrated that dyslipidemia was more frequent in the tinnitus group (*p*-value of 0.003), while the presence of diabetes mellitus, hypothyroidism, noise exposure, caffeine consumption, and smoking was similar in both groups. When considering the coexistence of arterial hypertension and comorbidities and habits, the association of arterial hypertension and caffeine consumption greater than 150 milliliters per day was more frequent in the tinnitus group (*p* < 0.0001).

**Table 4** shows data about the antihypertensive drugs used in both groups.

**Table 5** shows data from the numerical variables for the tinnitus' patients subgroups, those with and without arterial hypertension.

There was no statistical difference between the two subgroups concerning gender and race. Diabetes mellitus and dyslipidemia were more frequent in the subgroup with tinnitus and arterial hypertension (*p* = 0.017 and 0.02, respectively), while there were no differences concerning hypothyroidism, noise exposure, caffeine consumption, or smoking. Hearing loss was considered to be more prevalent in the subgroup with arterial hypertension (89.1%), although it was also frequent in the normotensive subgroup (75%, *p* = 0.032). No differences concerning the type or curve were found.

#### Table 3 | Prevalence of hearing loss among tinnitus and non-tinnitus patients (**χ**2 test).

Table 4 | Analysis of the categorical variable – antihypertensive drugs used according to the groups.

χ*-squared or Fisher tests.*

*ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; CCA, calcium channel antagonist.*

Figueiredo et al. Tinnitus and Hypertension

Table 5 | Analysis of the numerical variables (age, caffeine consumption, tinnitus duration, Visual Analog Scale, Tinnitus Handicap Inventory, Pitch Masking, and Minimum Masking Level) according to the subgroups of tinnitus patients (with and without arterial hypertension).

*Nineteen patients reported no caffeine consumption, nine patients could not estimate the duration of their tinnitus, and one patient could not report VAS and THI scores. IQA, interquartilic amplitude: Q1–Q3.*

*Bold is to highlight the significant data.*

*a Mann–Whitney test.*

*VAS, Visual Analog Scale; THI, Tinnitus Handicap Inventory; PM, Pitch Masking; MML, Minimum Masking Level; RE, right ear; LE, left ear; dB, decibel; dB SL, decibel Sensation Level.*

**Table 6** shows the tinnitus characteristics in both subgroups.

The most frequently described types of tinnitus were wheezing, whistle, and insect with no differences between the subgroups. The presence of multiple types of sound in the same patient was more frequently found in the hypertensive subgroup (*p* = 0.014). The prevalence of vascular tinnitus was 6.3% in the hypertensive subgroup and 1.3% in the normotensive one (*p* = 0.12) with muscular tinnitus being found in only two cases, both being in normotensive patients.

No significant differences were found between the subgroups concerning the otolaryngologic exam, including evaluation of the temporomandibular joint. Finally, there was no correlation discovered between the duration of arterial hypertension and tinnitus according to the Spearman coefficient (*p* = 0.77).

### DISCUSSION

Association studies are key sources of information for the comprehension of how one disorder may affect another. A study on Table 6 | Analysis of the categorical variables (tinnitus clinical characteristics) according to the tinnitus' patients' subgroups (with and without arterial hypertension).

χ*2 or Fisher test.*

*a*

*RE, right ear; LE, left ear.*

attempt to establish a correlation between arterial hypertension and tinnitus studies will help to elucidate characteristic in common that could be addressed in the diagnosis and therapeutic interventions. However, causal correlate studies would demand a plethora of different cases, including patients without tinnitus or arterial hypertension who were also free from other infirmities or habits which could be linked to tinnitus generation. Oftentimes, this elaborate type of study requires an extensive time period and patients with incredibly varied conditions.

A systematic review on this subject (24) argued that studies which have evaluated the prevalence of tinnitus among hypertensive patients failed to demonstrate an association (26–29). However, data from this study, along with other studies (2, 30, 31), demonstrated an association between tinnitus and arterial hypertension (hypertension prevalence in tinnitus patients = 44.4% against 31.4% in patients without tinnitus, *p* = 0.024).

The information above may lead one to infer that tinnitus could be a causal factor for hypertension, albeit it seems more reasonable to believe arterial hypertension is more a cofactor than a main cause of tinnitus.

Regarding hearing loss, the results are in conformity with prior studies, which found a high prevalence among tinnitus patients, as well as the higher prevalence of sensorineural pattern with descending configuration curves (2, 5).

Once hearing loss was added to (adjusted for) the statistical analysis, the association between tinnitus and arterial hypertension was no longer positive, suggesting that arterial hypertension may be a cause of hearing loss, which is related to most cases of tinnitus, as previously reported (1, 6). Having said so, if we think about tinnitus prevention, arterial hypertension may still be regarded as a possible important causal factor.

Arterial hypertension may affect the inner ear microcirculation, and it is known that comorbidities, such as diabetes mellitus and dyslipidemia, may enhance vascular impairment due to hypertension (10). Although dyslipidemia was more prevalent in the tinnitus group, there was no difference concerning the concomitancy of arterial hypertension and dyslipidemia. Diabetes mellitus also affects the inner ear microcirculation, but it may also have direct metabolic effects on cochlea (4, 5, 20). There was a statistical tendency in favor of a higher prevalence of the concomitancy of diabetes and hypertension in the tinnitus group. As for hypothyroidism, another metabolic disease frequently cited as related to tinnitus (6), no differences between the two groups were found, either as an isolated factor or in association with arterial hypertension. The comparison between the tinnitus subgroups (with and without hypertension) demonstrated that diabetes mellitus and dyslipidemia were more prevalent in hypertensive tinnitus patients, suggesting that these diseases may act synergically upon the generation, maintenance and/or aggravation of tinnitus.

The excessive consumption of caffeine is believed to have a negative impact on hypertension control (32), but its association with tinnitus has been doubted by recent studies (9, 33, 34). Data from this study failed to demonstrate any association between tinnitus and caffeine consumption. In fact, the concomitancy of hypertension and caffeine intake greater than 150 ml per day was more prevalent in the control group, which may reflect the widespread patients' concept that caffeine worsens tinnitus. Smoking, which is believed to worsen both tinnitus and hypertension, probably by impairing macro and microcirculation (10, 35), was more prevalent in the tinnitus group, either as an isolated factor or concomitant to hypertension.

Considering these findings, one might speculate that vascular changes, which may affect cochlear microcirculation, leading to hair cells damage and, consequently, tinnitus is probably a common pathophysiological scenario for many conditions, such as arterial hypertension, dyslipidemia, diabetes mellitus, smoking, and caffeine abuse. These conditions are not infrequently found in a single patient and may act sinergically, multiplying the damage to the auditory system.

Although noise exposure is considered as one of the main causes of tinnitus (4, 6), data from this study failed to establish an association between noise exposure and tinnitus, either isolated or concomitant to hypertension. This finding should be considered with caution, being that the study was performed in an industrial city where many workers are exposed to industrial or recreational noise.

The average age of patients with tinnitus and arterial hypertension was significantly higher than the age of those in the group with tinnitus and no arterial hypertension. In the hypertension tinnitus subgroup most of the patients were 60 years or older, while the opposite was verified in the subgroup without hypertension. Both tinnitus and arterial hypertension are more prevalent in the elderly (2, 10), but these findings may also be due to some synergistic action of presbycusis and arterial hypertension contributing to tinnitus generation and, eventually, aggravation.

The difference concerning arterial hypertension duration, which was significantly lower in the tinnitus group, may be due to a lack of proper control of blood pressure in the first years, which may lead to perfusion and reperfusion vascular events in the cochlea. More studies are needed to clarify these findings.

The median of the pitch masking was higher (6 kHz) in the subgroup without hypertension than when compared to the hypertension subgroup (4 kHz), although this difference was not statistically significant. Both measures are in agreement with most of the references, tinnitus ranging from 3 to 8 kHz, which corresponds to the usually most affected frequencies in hearing loss (36, 37). As for the MML, the averages of both subgroups were the same (15 dB SL), which is somewhat higher than the usually reported sensation level of 5–10 dB SL (37). No differences were demonstrated concerning the degree of intensity and distress due to tinnitus in both subgroups, considering the VAS and the THI.

The tinnitus' characteristics were similar in both groups, including the type of sound perceived by the patients. The fact that multiple sounds' tinnitus was more prevalent in the hypertensive patients may reflect the multicausality of tinnitus, hypertension being one of the possible factors involved.

Although the average duration of arterial hypertension was longer than the duration of tinnitus and for most of the patients the onset of hypertension preceded the onset of tinnitus, the methodology of this case–control does not allow the conclusion that hypertension is a causal factor for tinnitus.

The ototoxicity of many antihypertension medications has been well established, especially with diuretics (21, 22). Data from this study demonstrated that the use of ACE inhibitors, thiazidic diuretics, potassium-sparing diuretics, and calcium channels blockers was more prevalent in the tinnitus hypertensive patients than in the control group. These findings have a partial correspondence with prior studies (16) and, although they are not strong enough to justify a correlation between an eventual ototoxicity of these drugs and the presence of tinnitus (for example, multidrug therapy for hypertension is very frequent), it appears that further, more detailed studies on this subject should be performed.

### CONCLUSION

There is an association between tinnitus and arterial hypertension. This association is particularly strong in older patients and cannot be dissociated from the hearing loss, which was also more prevalent among tinnitus patients. The use of thiazidic and potassium-sparing diuretics, ACE inhibitors, and calcium channels blockers was more prevalent in tinnitus patients.

The clinical and psychoacoustic characteristics of tinnitus in hypertensive and normotensive patients were similar, as well as tinnitus-related distress.

### AUTHOR CONTRIBUTIONS

RF – study design, data collection, and writing. AA – data collection. NP – study design and writing.

#### ACKNOWLEDGMENTS

Rosângela Martins, for the statistics. Lisa Morrison Thuler, for the manuscript English revision.

### FUNDING

This work was supported by a grant from CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Governo Federal do Brasil.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Figueiredo, Azevedo and Penido. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Innovations in Doctoral Training and Research on Tinnitus: The European School on Interdisciplinary Tinnitus Research (ESIT) Perspective

Winfried Schlee1†, Deborah A. Hall 2, 3†, Barbara Canlon4 , Rilana F. F. Cima5 , Emile de Kleine6 , Franz Hauck 7 , Alex Huber 8 , Silvano Gallus 9 , Tobias Kleinjung8 , Theodore Kypraios 2 , Berthold Langguth1 , José A. Lopez-Escamez 10, 11, Alessandra Lugo9 , Martin Meyer 12, Marzena Mielczarek 13, Arnaud Norena14, Flurin Pfiffner 8 , Rüdiger C. Pryss 15, Manfred Reichert 15, Teresa Requena10, Martin Schecklmann1 , Pim van Dijk 6 , Paul van de Heyning16, Nathan Weisz 17 and Christopher R. Cederroth4 \*

#### Edited by:

Aurel Popa-Wagner, University of Rostock, Germany

#### Reviewed by:

Leslie Samuel Smith, University of Stirling, United Kingdom Jae-Jin Song, Seoul National University Bundang Hospital, South Korea

#### \*Correspondence:

Christopher R. Cederroth<EMAIL_ADDRESS>
> † Shared first authors.

Received: 31 July 2017 Accepted: 29 December 2017 Published: 12 January 2018

#### Citation:

Schlee W, Hall DA, Canlon B, Cima RFF, de Kleine E, Hauck F, Huber A, Gallus S, Kleinjung T, Kypraios T, Langguth B, Lopez-Escamez JA, Lugo A, Meyer M, Mielczarek M, Norena A, Pfiffner F, Pryss RC, Reichert M, Requena T, Schecklmann M, van Dijk P, van de Heyning P, Weisz N and Cederroth CR (2018) Innovations in Doctoral Training and Research on Tinnitus: The European School on Interdisciplinary Tinnitus Research (ESIT) Perspective. Front. Aging Neurosci. 9:447. doi: 10.3389/fnagi.2017.00447 1 Department of Psychiatry and Psychotherapy of the University of Regensburg at Bezirksklinikum Regensburg, University of Regensburg, Regensburg, Germany, 2 NIHR Nottingham Hearing Biomedical Research Centre, Nottingham, United Kingdom, 3 Otology and Hearing Group, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, Nottingham, United Kingdom, 4 Section of Experimental Audiology, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 5 Clinical Psychological Science, Faculty of Psychology and Neuroscience, Maastricht University, Maastricht, Netherlands, 6 Department of Otorhinolaryngology/Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen, Netherlands, 7 Institute of Distributed Systems, Ulm University, Ulm, Germany, 8 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Zurich, University of Zurich, Zurich, Switzerland, 9 Department of Environmental Health Sciences, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy, 10 Otology and Neurotology Group, Department of Genomic Medicine, Centro Pfizer – Universidad de Granada – Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Granada, Spain, 11 Department of Otolaryngology, Instituto de Investigación Biosanitaria ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Granada, Spain, 12 Neuroplasticity and Learning in the Healthy Aging Brain (HAB LAB), Department of Psychology, University of Zurich, Zurich, Switzerland, 13 Department of Otolaryngology, Laryngological Oncology, Audiology, and Phoniatrics, Medical University of Lodz, Lodz, Poland, 14 Centre National de la Recherche Scientifique, Aix-Marseille University, Marseille, France, 15 Institute of Databases and Information Systems, Ulm University, Ulm, Germany, 16 Department of ORL and Head and Neck Surgery, Antwerp University Hospital, University of Antwerp, Antwerp, Belgium, 17 Division of Physiological Psychology, Centre for Cognitive Neuroscience, University of Salzburg, Salzburg, Austria

Tinnitus is a common medical condition which interfaces many different disciplines, yet it is not a priority for any individual discipline. A change in its scientific understanding and clinical management requires a shift toward multidisciplinary cooperation, not only in research but also in training. The European School for Interdisciplinary Tinnitus research (ESIT) brings together a unique multidisciplinary consortium of clinical practitioners, academic researchers, commercial partners, patient organizations, and public health experts to conduct innovative research and train the next generation of tinnitus researchers. ESIT supports fundamental science and clinical research projects in order to: (1) advancing new treatment solutions for tinnitus, (2) improving existing treatment paradigms, (3) developing innovative research methods, (4) performing genetic studies on, (5) collecting epidemiological data to create new knowledge about prevalence and risk factors, (6) establishing a pan-European data resource. All research projects involve inter-sectoral partnerships through practical training, quite unlike anything that can be offered by any single university alone. Likewise, the postgraduate training curriculum fosters a deep knowledge about tinnitus whilst nurturing transferable competencies in personal qualities and approaches needed to be an effective researcher, knowledge of the standards, requirements and professionalism to do research, and skills to work with others and to ensure the wider impact of research. ESIT is the seed for future generations of creative, entrepreneurial, and innovative researchers, trained to master the upcoming challenges in the tinnitus field, to implement sustained changes in prevention and clinical management of tinnitus, and to shape doctoral education in tinnitus for the future.

Keywords: tinnitus, education, medical, hearing, PhD studentship, heterogeneity of tinnitus

#### BACKGROUND

Tinnitus is a condition associated with a continuous auditory percept in the ears or head and can arise as a symptom of many different medical disorders. Assuming a conservative tinnitus prevalence of 10% (1% of severe tinnitus; McCormack et al., 2016) for the 425 million adults living within the European Union (EU), tinnitus affects more than 42 million citizens and is experienced as a severe problem by more than 4 million. Moreover, incidence of new cases is expected to grow over the next few decades (Nondahl et al., 2002, 2010). Although much progress has been made in understanding the pathophysiology (Langguth et al., 2013; Elgoyhen et al., 2015), tinnitus remains a scientific and clinical enigma (Baguley et al., 2013). Unfortunately, tinnitus remains an unmet clinical need and complaining patients are often told "to live with it" (Cederroth et al., 2013). The condition is very common and of varying severity, but the fundamental mechanisms of tinnitus are still incompletely understood. Although not all individuals are unduly troubled, many find the disorder life-changing. In cases with severe tinnitus, mental disorders, and symptoms such as anxiety, depression, insomnia, and concentration problems can impair quality of life often to a level that leads to sick leave and disability pension (Friberg et al., 2012). Therefore, severe tinnitus contributes to a substantial cost to health care and to society at large. In the Netherlands alone, the economic burden of tinnitus is estimated at up to e10.8 billion, with the greater impact being related to socio-economic factors (Maes et al., 2013). The health care costs are enormous. In England there are 750,000 medical consultations yearly with the primary complaint of tinnitus (El-Shunnar et al., 2011).

There is no licensed pharmacological therapy and there is little high quality evidence for the success of palliative management strategies. The Cochrane Library currently lists 9 completed systematic reviews on different tinnitus treatments; namely Tinnitus Retraining Therapy (TRT), Cognitive Behavioral Therapy (CBT), hyperbaric oxygen therapy, sound therapy (masking), hearing aids, repetitive transcranial magnetic stimulation (rTMS), ginkgo biloba, anticonvulsants, and antidepressants (Phillips and McFerran, 2010; Hoekstra et al., 2011; Meng et al., 2011; Baldo et al., 2012; Bennett et al., 2012; Hobson et al., 2012; Hilton et al., 2013; Espinosa-Sánchez et al., 2014; Hoare et al., 2014; Person et al., 2016). However, no uniformly effective treatment for tinnitus has yet been identified. Two main reasons have been discussed: (1) methodological limitations in study design with a paucity of Randomized Controlled Trials (RCTs) and no consensus about what and how to measure therapeutic outcome (Hall et al., 2016), and (2) a large heterogeneity in the patient population with respect to etiology, genetic, and clinical phenotype (Elgoyhen et al., 2015).

Tinnitus is a symptom rather than a distinct disease, and its multivariate manifestations can be subtyped according to various dimensions such as its etiology, time since onset, perceptual characteristics (i.e., pitch, loudness, location, and temporal dynamics), perceived emotional distress, and comorbidities. Progress in scientific understanding and clinical management needs to first address this heterogeneity by identifying scientifically and clinically meaningful subtypes of the condition. Subtyping can then guide the definition of relevant inclusion/exclusion criteria in clinical research and of stratification variables for allocating patients to different intervention groups in RCTS. Subtyping can also guide more sophisticated methods of multivariate data analysis than have been applied hitherto. Current knowledge about etiology, perceptual characteristics, and the neurobiological correlates of tinnitus is not sufficient to enable effective subtyping (Lopez-Escamez et al., 2016). These problems are indicating the need for new approaches.

### AN INTERDISCIPLINARY APPROACH IS CRITICAL

Across the EU, no single healthcare system, research organization, or commercial enterprise has an adequate coverage of all relevant issues related to tinnitus and this has resulted in a patchwork of approaches without any coherent framework. Clinically speaking, tinnitus is managed by a variety of different practitioners including general practitioners, otologists, audiologists, psychologists, psychiatrists, neurologists, physical therapists, and dentists (Hall et al., 2011). Academically speaking, tinnitus is of interest to animal neurophysiologists, neuroscientists, epidemiologists, geneticists, trialists, biostatisticians, biomedical engineers, software engineers, and data mining experts. The European School on Interdisciplinary Tinnitus Research (ESIT) uniquely fosters an environment where knowledge and ideas are shared beyond current sectoral borders. The ESIT project provides an international interdisciplinary network of experts from these relevant disciplines working toward a coordinated approach to tinnitus research. Such a partnership is an essential part of the project. It will support data sharing and meta-analyses to gain new insights about tinnitus, to develop an evidence-based treatment protocol for effective personalized medicine and to identify new innovative treatment approaches.

# ESIT MANAGEMENT AND PROJECT DETAILS

The ESIT project started on April 1, 2017 with an overall duration of 4 years. The 15 PhD start their work in October 2017. Each student receives funding for 3 years to undertake their PhD. In **Figure 1** we outline the work packages of the ESIT project. The first three work packages focus on the scientific goals, while work packages 4–7 concentrate on the effective management of the project (i.e., governance, recruitment and training, communications, and longer-term sustainability). The ESIT office is led by Schlee and based at the department of psychiatry and psychotherapy at the University Clinic Regensburg. More details about the ESIT project and the most recent achievements of the project can be found on the project website (www.esit. tinnitusresearch.net).

#### ESIT's INNOVATIVE PERSPECTIVE ON TINNITUS RESEARCH

ESIT is an EU-funded Marie Skłodowska-Curie Innovative Training Network with 12 top-level research institutions in 10 European countries, supporting 15 PhD projects. The research performed under ESIT will be geared toward a more personalized medicine approach, with improved diagnosis and selection of the best-suited therapy based on the individual patient profile. The 15 PhD projects will collectively address three major objectives that are managed under three research-specific work packages. In total, 2 clinics, 8 commercial enterprises, 2 patient organizations, 5 partner academic institutions, and 2 non-profit organizations are actively partnering in the projects and will support 2–3 month research secondments. Secondments are research visits or practical trainings with other academic institutions or industry partners to promote inter-sectoral exchange and embed a broader perspective into the research collaboration from the outset. Commercial partners will also share technological innovations for research purposes.

**Research-specific work package 1. "meaningful individual differences"** Co-ordinated by Lopez-Escamez, work package 1 seeks to determine meaningful individual differences in tinnitus by integrating knowledge and experience from all relevant clinical and scientific disciplines, and combining it with patient-centered and commercial perspectives. This will be achieved through interdisciplinary ESIT partnerships with the Meniere's Disease Society (patient organization), Tinnitus Research Initiative Foundation (non-profit research organization), Sensorion Pharmaceuticals (commercial sector), Julius Maximilian's University Würzburg, and the Knowledge Management and Discovery Laboratory at the Otto von Guericke University. Through this work package:

**Research-specific work package 2. "novel personalized treatment solutions"** Co-ordinated by Weisz, work package 2 seeks to develop novel personalized treatment solutions that respect each patient profile and integrate them with state-ofthe-art technological innovations. This will be achieved through interdisciplinary ESIT partnerships with Sivantos, Cochlear Europe, Bee Group AG and Soterix Medical Inc. (commercial sector), and Del Bo Technologia per l'ascolto (independent clinic). Through this work package:

**Research-specific work package 3. "comparability of scientific and clinical results"** Co-ordinated by Gallus, work package 3 seeks to promote international comparability of findings across all major relevant disciplines. This will be achieved through interdisciplinary ESIT partnerships with the British Tinnitus Association (patient organization), Brain Products (commercial sector), Nottingham University Hospitals NHS Trust (clinic), and the Institute of Computer Science, University of Tartu. Through this work package:

Stockholm, and University of Regensburg, respectively.

This brief overview of the research projects illustrates how the ESIT consortium is characterized by interdisciplinary partnerships both within and across research-specific work packages 1–3. Many projects benefit from the exchange of measurement tools, data sharing, intersectoral knowledge transfer, and dissemination. The overall research program is designed to promote synergies between projects.

# ESIT's INNOVATIVE PERSPECTIVE ON TINNITUS TRAINING

ESIT provides the first specialized doctoral curriculum on the topic of tinnitus. Acknowledging that "twenty-first century skills" require more than just knowledge building, ESIT PhD students will develop a balance of transferable competencies, as well as tinnitus-specific knowledge and skills (news feature 2015). In this sense it is unique. Each ESIT PhD student will actively manage his/her own dynamic Personal Career Development Plan. To do this we will promote the Vitae Researcher Development Framework Planner, which is a web-based application for mapping professional development (Vitae, 2017). The planner can be used to keep a unified record of all professional development activities, identify their current expertise and capabilities, record learning and development goals and monitor progress, and upload files such as CVs, conference details, and testimonials to record their personal achievements for lifelong learning. The planner uses the Vitae Researcher Development Framework, which considers four essential domains of learning and development and subdomains which inform the learning objectives of the ESIT training modules (**Figure 2**).

FIGURE 2 | Overview of the ESIT training curriculum structured according to the Vitae Researcher Development Framework. The four essential domains of learning and development are given in the four squares, with individual training modules arranged below.

ESIT offers an innovative doctoral training program (see **Figure 2**) for ambitious young researchers and provides those PhD students with a set of unique academic, clinical, charity, and commercial sector experiences as well as learning opportunities that extend far beyond any typical academic training program. In the same way that all beneficiaries and most partner organizations are contributing to the research projects in work packages 1–3, work package 5 co-ordinated by Hall describes the training curriculum and the input from many of the ESIT consortium members. In total, 10 commercial enterprises, 6 patient and non-profit organizations, 6 partner academic institutions, 1 health authority and 11 independent clinics will provide practical training for the 15 ESIT students. Most training will be provided by ESIT training schools, satellite events at annual conferences and internet-based courses. Self-study will be guided by supervisors and coordinated and monitored by a Supervisory Board.

A prerequisite for impactful research is an awareness of realworld challenges in the tinnitus clinic. At the start of the program, all ESIT PhD students will complete a clinical internship whereby they will experience for 1 week the activities within a tinnitus clinic in a European country. They will meet with many tinnitus patients and tinnitus care providers, develop an appreciation of the symptoms and impact of tinnitus on the lives of real people, learn to understand the care system in the respective country, and critically evaluate the challenges in that system.

# PLANNED MAJOR DISSEMINATION ACTIVITIES

Real-world development opportunities are afforded through several major dissemination activities. First, ESIT PhD students will write a special commissioned feature for publication in "ENT & Audiology News" magazine aimed at hearing healthcare professionals. Second, the ESIT network aims for a completely revised and updated 2nd edition of the "Textbook of Tinnitus" (Møller et al., 2011). The revised edition will be tailored toward becoming the number one choice as a textbook for doctoral and medical training in tinnitus. Other dissemination activities include an updated version of the Wikipedia page for "Tinnitus" (Wikipedia, 2017Q12), an educational video on tinnitus prepared in the major European languages, and dissemination of activities through social media channels. Dissemination will be coordinated by Cederroth, leader of the work package 6.

#### IMPACT ON CAREER PROSPECTS

To enable clinical relevance of academic research and its rapid translation into improved tinnitus healthcare, there is an urgent need for closer intersectoral collaboration. In conclusion, ESIT will achieve this by providing high-level training with a multidisciplinary supervisory team. The program will equip ESIT PhD students not only with a comprehensive understanding of the challenges faced by healthcare providers in treating tinnitus and by industry in providing technological healthcare solutions, but also to lay the foundations for leadership positions in the relevant academic and non-academic sectors. The networkintegrated training program will ensure that the ESIT PhD student can effectively communicate between disciplines and will have the skills to identify synergistic opportunities, to build bridges between disciplines to exploit these connections, and to interact successfully with the private sector. Exposure across disciplines and sectors is unique and is essential to advance ESIT

### REFERENCES

student personal career development and lead the students into European leadership positions in the future. In this way, ESIT will create a new generation of tinnitus experts who are sensitive to the issues of heterogeneity, have a broad knowledge base and have first-hand experience of the possibilities for inter-sectoral disciplines.

# AUTHOR CONTRIBUTIONS

WS, DH, and CC drafted the initial version of the manuscript and created the figures. All authors contributed to the development of the ESIT project and contributed equally to all other stages of the manuscript development. All authors approved the manuscript.

### FUNDING

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722046.

### ACKNOWLEDGMENTS