Resting-state Functional MRI Study of Mice with Tinnitus Induced by Traumatic Noise Exposure and Sodium Salicylate Overdose

Resting-state Functional MRI Study of Mice with Tinnitus Induced by Traumatic Noise Exposure and Sodium Salicylate Overdose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Resting-state Functional MRI Study of Mice with Tinnitus Induced by Traumatic Noise Exposure and Sodium Salicylate Overdose Weixun Zhang, Jianan Shi, Yuwen Zhang, Rui Ma, Meirong Chen, Juanmei Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5014033/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tinnitus, a phantom auditory sensation, significantly impacts quality of life and social interactions. While central gain changes, including hyperactivity and increased neural synchrony, have been implicated in tinnitus, the underlying neural mechanisms remain unclear. This study utilized resting-state functional magnetic resonance imaging (rs-fMRI) to investigate the neural basis of tinnitus induced in mice by two distinct methods, traumatic noise exposure and sodium salicylate overdose. Behavioral tests confirmed the successful induction of tinnitus in both groups. rs-fMRI analysis revealed distinct patterns of neural activity in mice with tinnitus compared with control mice. Traumatic noise exposure induced an increase in amplitude of low-frequency fluctuations (ALFF) in the paraflocculus and auditory cortex, as well as a decrease in regional homogeneity (ReHo) in limbic system regions. On the other hand, sodium salicylate overdose led to decreased ALFF and ReHo in the auditory cortex, somatosensory system and limbic system regions. Functional connectivity analysis further highlighted differences between the two models, with traumatic noise exposure affecting connectivity with the inferior colliculus and sodium salicylate overdose affecting connectivity with the medial geniculate body. These findings suggest that traumatic noise exposure and sodium salicylate overdose induce tinnitus through distinct neural mechanisms, potentially involving different neural circuits and pathways. Further research is needed to explore these mechanisms and develop targeted therapies for tinnitus. Functional MRI Noise Sodium Salicylate Tinnitus auditory cortex medial geniculate body Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Tinnitus, a phantom auditory sensation that dramatically and negatively affects an individual’s quality of life and social interactions, affects approximately 10–15% of the general population. Severe tinnitus affects approximately 1–2% of the global population (Langguth et al. 2013; Auerbach et al. 2014). Unfortunately, there is currently no widely effective therapy that eliminates tinnitus, perhaps because its neural basis remains elusive. Most cases of chronic tinnitus are associated with central gain changes, including neuronal hyperactivity, bursting discharges and increased cortical neural synchrony in the auditory system, which occurs after deafferentation of auditory nerves (Adjamian et al. 2014; Turrigiano 2012). Predictive brain frameworks suggest that changes in cortical homeostatic plasticity in response to central gain changes are part of the pathogenesis of tinnitus (Noreña and Farley 2013). ; the role of these changes in tinnitus pathogenesis is still unknown but may result from a lack of inhibition in the central auditory pathway. T The use of animal models to study tinnitus is highly valuable, as these models allow for strict experimental control that is not permissible in human research (Henton and Tzounopoulos 2021). The systemic application of an overdose of sodium salicylate (SS) and traumatic noise exposure are two commonly used methods for inducing tinnitus in animals. Although both salicylate and traumatic noise can produce tinnitus symptoms that are similar to those of chronic tinnitus patients, the underlying mechanisms are likely different. The blood oxygen level-dependent (BOLD) signal observed on magnetic resonance imaging (MRI) measures neuronal activity using the ratio of oxyhemoglobin to deoxyhemoglobin as a contrast mechanism (Leuthardt et al. 2015). The accumulation of spontaneous, highly synchronized, short time-lag neural activations between two groups of neurons in two separate regions is reflected in a positively correlated, homogeneous hemodynamic BOLD signal obtained during resting-state functional MRI (fMRI)(Cardin et al. 2023). A functional association network of executive functions and cognitive control has been successfully constructed using resting-state functional connectivity (FC) (Cohen and D'Esposito 2016). The goal of our study was to map the regions of neural hyperactivity and enhanced FC that characterize the central gain changes in mice with tinnitus induced by SS overdose and traumatic noise exposure. Behavioral task performance was used to identify the characteristic tinnitus frequencies in the mice. We subsequently used rs-fMRI to explore potential changes in the fractional amplitude of low-frequency fluctuations (fALFF) and FC of the core brain regions in the auditory pathway. Our results demonstrated that the medial geniculate body (MGB) and auditory cortex (AC) had the highest degree of hypersensitivity in the tinnitus mouse model, indicating the potential vital role of the MGB in the pathology of tinnitus. Methods Animals Adult CBA/Ca mice (n=37) weighing ~25 g and aged ~6 weeks at the start of the experiments were used in this study. The mice were housed 3–5 animals per cage within a colony room with a 12-h light‒5-h dark cycle at 23°C. All animal procedures were approved by the Ethics Committee of the Eye Ear Nose & Throat Hospital of Fudan University. Traumatic noise exposure The mice assigned to the noise exposure group were placed in custom-made metal cages before being moved to soundproof boxes containing four speakers for noise exposure. The difference between the noise sound pressure level received by the mouse's ears and the target sound pressure level (SPL) was less than 1 dB. Noise was generated by a TDT System 3 (TDT, USA) , amplified through a P9500S Power Amplifier (YAMAHA, Japan), and played through 4 Pyramid Tweeter TW67 speakers (Pyramid, USA). The parameters of the noise exposure protocol were 8–16 kHz narrowband noise at an intensity of 100 dB SPL for 2 hours. Overdose sodium salicylate SS (Sigma‒Aldrich, USA) was administered to mice in the ototoxic drug overdose group through intraperitoneal (i.p.) injection. Previous studies have shown that even through systemic administration, SS can pass through the blood–brain barrier, increasing the SS concentration in cerebrospinal fluid [11, 12]. Studies have also shown that 250~400 mg/kg is an effective concentration depending on the animal species [13]. In this study, we chose 350 mg/kg i.p. injection for 1 week to induce tinnitus. Injections were performed at approximately the same time every day. Auditory brainstem responses (ABRs) test The hearing thresholds of animals exposed to noise and ototoxic drugs were measured, and the ABRs were recorded. The animals were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and placed in a soundproof box (Tucker-Davis Technologies, USA) before testing. Five frequencies (4, 8, 16, 24 and 32 kHz) were tested using a small speaker placed 10 cm from the midpoint of the two ears (MF1 multifield magnetic speakers and P9500S YAMAHA power amplifier), and the results were recorded. The waveforms were visualized and recorded using BioSigRZ software and a TDT System 3 data acquisition system (Tucker-Davis Technologies, USA). The sound stimulus started at a 90 dB SPL and was decreased by 5 dB until the hearing threshold was determined. For each SPL, 600 responses were recorded and averaged. Offline analysis of stored waveforms was used to determine ABR wave 1 thresholds. The hearing threshold was defined as the lowest stimulus level that elicited repeatable wave 1 responses on visual inspection of stacked waveforms. Behavioral tests of tinnitus Prepulse inhibition of the acoustic startle reflex (PPIAS) and gap prepulse inhibition of the acoustic startle reflex (GPIAS) were tested in all the mice before tinnitus induction and 7 d after SS injection or 14 d after noise exposure using a custom-built system. All behavioral tests were conducted in a sound-attenuating cubicle. The sounds were presented through a standard acoustic startle sound speaker (MED Associates, USA), which was positioned in front of the animals. A narrow bandpass-filtered background sound (1 kHz bandwidth centered at 10, 16, 20, 24 and 32 kHz) was presented at 60 dB SPL for 17~25 s (randomly varied) before the presentation of an acoustic startle stimulus (white noise, 115 dB SPL, 20 ms). The entire session consisted of 15 startle-only trials, 15 broadband noise (BBN) trials and 10 gap detection trials for each frequency. Gap detection trials were identical to startle-only trials except that a 50-ms silent gap was inserted 130 ms before the startle stimulus. For each frequency, 10 gap/no-gap ratios were collected and averaged, and the gap detection ability was determined using the gap detection ratio, calculated as the ratio of the startle response waveform peak value with gaps to the peak value without gaps. For a given frequency, a gap startle ratio closer to 0 indicated a greater GPIAS, and a higher gap startle ratio indicated a defect in gap detection. The PPIAS is essentially the inverse of the GPIAS, except that the 50-ms silent gaps in the GPIAS were replaced with a 70 dB SPL, 50 ms prepulse with different frequencies (8, 10, 16, 20, 24, 32 kHz and BBN). The calculation for the PPIAS prepulse detection ratio is identical to that of the GPIAS gap detection ratio; frequencies with a ∆ratio of ≥0.2 were called tinnitus frequencies. Mice with the tinnitus frequency were considered tinnitus mice. Resting state functional magnetic resonance imaging (rs-fMRI) scanning MRI scanning was carried out at the Zhangjiang Brain Imaging Centre. Each mouse was placed in an anesthesia box with isoflurane before the test (induction concentration, 5%; maintenance concentration, 0.5%) (Zoetic, USA). To simulate the blood flow of awake animals, dexmedetomidine (Zoetic, USA) was administered by i.p. injection into each mouse (0.03 mg/kg for embolism, 0.015 mg/kg for continuous subcutaneous injection). During the image acquisition procedures, the respiratory rate of each mouse was maintained between 40–60 breaths/min; the rectal temperature was maintained at 36°C–37°C; the oxygen saturation was maintained at 98–100%; and the heart rate was maintained between 200–300 beats/min. MRI data were obtained on a 7.0-T animal MRI scanner (Bruker BioSpec 94/30, Ettlingen, Germany). For functional imaging, 30 axial slices were acquired using gradient-echo echo-planar imaging (GE-EPI) with the following parameters: repetition time (TR) = 2 s, echo time (TE) = 15 ms, matrix size = 80 × 67, field of view (FOV) = 30 × 30 mm 2 , and voxel size = 0.375 × 0.373 × 0.6 mm 3 . High-resolution anatomical T2-weighted fast spin‒echo images were acquired at the same slice location with the following parameters: TR/TE = 3735 ms/33 ms, matrix = 256 × 256, FOV = 30 × 30 mm 2 , and average = 1. MRI data processing and statistical analysis All preprocessing and data analysis were performed with Statistical Parametric Mapping (SPM12) software (Welcome Department of Imaging Science; http://www.fil.ion.ucl.ac.uk/spm) and DPARSF (http://rfmri.org/DPARSF) according to standard procedures, including removal of the first 10 timepoints, slice timing, realignment, spatial normalization, smoothing, removal of the linear trend and ALFF calculation. Voxelwise statistical analysis was performed using the framework of a general linear model (GLM). To identify significant differences between groups, a two-sample t test was carried out using SPM12, and brain regions with significant ALFF and regional homogeneity (ReHo) changes were identified according to a voxel-level height threshold pf p < 0.001. For the resting-state analysis, 7 main brain regions on the auditory pathway were selected as regions of interest (ROIs), including the AC, dorsal cochlear nucleus (DCN), inferior colliculus (IC), MGB, nucleus of the lateral lemniscus (NL), superior olivary body (SOB), and ventral cochlear nucleus (VCN). FC was calculated using correlation analysis among the ROIs, and time courses from the average signal in each ROI were obtained. Two-sample t tests were used to compare differences in connectivity between the experimental groups and the control group. A P value less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7 software (Prism 7; GraphPad Software Inc., La Jolla, CA, USA). Normally distributed continuous variables are presented as the means ± standard errors of the means (SEMs) or means ± standard deviations (SDs). Unless otherwise noted, the level of significance is illustrated in the figures with the following symbols or shaded areas (not significant (n.s.): p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.001). Fig.1. Experimental timeline. The animals were randomly assigned to three groups: the control group, the traumatic noise exposure group and the overdose sodium salicylate group. Baseline hearing levels (Day 0) were measured through ABRs in all groups. (A) The animals in the traumatic noise exposure group received 100dB SPL 8-16kHz narrowband noise exposure for 2 hours. ABRs evaluations were preformed in 1 day, 7 days, and 14 days after the traumatic noise exposure. Behavioral tests and rs-fMRI scan were only conducted 14 days after noise exposure. (B) In the overdose sodium salicylate group, the mice were given 350mg/kg sodium salicylate intraperitoneally once daily for 7 successive days. The mice were conducted ABRs, behavioral tests and rs-fMRI scan in 1 day after the end time point of sodium salicylate injection. Results SS injection produces a hearing threshold shift, and noise exposure produces a temporary hearing threshold shift ABR thresholds were analyzed to determine the degree of hearing loss in the mice in both the SS injection group and the noise exposure group. Tests were conducted on days 0 and 7 of continuous injection for the SS group and on days 0, 1, 7 and 14 after noise exposure for the noise exposure group. Compared with before injection, 7 days after SS injection, the hearing threshold of the mice (n=10) was significantly increased at almost all frequencies except 4 kHz ( Fig. 2A ). Similarly, we also found a significant threshold shift at days 1 and 7 in the noise exposure group. However, on day 14 after noise exposure, the increase in hearing threshold had reversed ( Fig. 2B ). Based on these results, we concluded that SS produced a permanent threshold shift (PTS), whereas noise exposure produced a temporary threshold shift (TTS). Fig.2. (A) Sodium salicylate injection produce long-term, significant hearing threshold shift. On day 7 of continuous injection, hearing thresholds at each frequency are 74.375±1.475 dB SPL (4kHz, P=0.1234), 45.000±1.637 dB SPL (8kHz, P=0.0019), 41.250±1.250 dB SPL (16kHz, P<0.0001), 46.875±1.315 dB SPL (24kHz, P<0.0001), and 55.625±1.990 dB SPL (32kHz, P<0.0001). (B) Noise exposure produce permanent, reversible hearing impairment. On day 1 right after noise exposure, hearing threshold was significantly elevated at all frequencies but recovered to normal on day 14 after noise exposure. A maximum threshold shift of over 30 dB was detected at 32 kHz at day 1 post-exposure and recovered to normal pre-exposure values by day 14. Mean ± SEM is shown in each group. Paired t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Establishment of a tinnitus mouse model by SS overdose and traumatic noise exposure To verify the effectiveness of SS overdose and traumatic noise exposure in inducing tinnitus in a mouse model, we conducted behavioral tests (PPIAS and GPIAS) in both experimental groups. The SS overdose group was tested 1 day after continuous injection, whereas the traumatic noise exposure group was tested 14 days after noise exposure. Mice that exhibited the tinnitus frequency in both the PPIAS and GPIAS tests were defined as tinnitus mice in our study. According to the PPIAS prepulse detection ratio and GPIAS gap detection ratio, we divided these mice into four groups: traumatic noise exposure with tinnitus (NE-Tinnitus (+)) group, traumatic noise exposure without tinnitus (NE-Tinnitus (-)) group, SS overdose group with tinnitus (SS-Tinnitus (+)) group and SS overdose without tinnitus (SS-Tinnitus (-)) group (Figs. 3 and 4) . Six mice (6/10) in the SS injection group and four (4/10) in the traumatic noise exposure group presented behavioral evidence of tinnitus. A comparison of the noise exposure group and the SS group revealed that the ΔGPIAS and ΔPPIAS ratios were significantly greater in mice with tinnitus than in those without tinnitus (Fig. 3F and Fig. 4F). Compared with the NE-Tinnitus (-) and control groups, the NE-Tinnitus (+) group had significantly greater ΔPPIAS and ΔGPIAS ratios (Fig. 3F). However, the average ΔGPIAS and ΔPPIAS ratios in the SS-Tinnitus (+) group were not different from those in the SS-Tinnitus (-) and control groups (Fig. 4F ). Although both traumatic noise exposure and SS overdose can cause tinnitus, the mice with tinnitus caused by traumatic noise exposure exhibited more interesting behavioral signs. Fig. 3. ∆PPIAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆PPIAS ratio were calculated on BBN, 8kHz, 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. (A-E) ∆PPIAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). (F) Comparison of average ∆PPIAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean ± SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p 0.05; #: p < 0.05; ##: p < 0.01; ###: p < 0.001; ####: p < 0.001 by two-way ANOVA. Fig. 4. ∆GAPAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆GAPAS ratio were calculated on 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. (A-E) ∆GAPAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). (F) Comparison of average ∆GAPAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean ± SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p 0.05; #: p < 0.05; ##: p < 0.01; ###: p < 0.001; ####: p < 0.001 by two-way ANOVA. Alterations in ALFF in the SS overdose and traumatic noise exposure groups From the perspective of energy metabolism, ALFF is defined as the density of neurons with spontaneous synchronized firing in the frequency range of 0.01~0.1 Hz; this value reflects brain region activity (Biswal B 1995; Cordes D 2000; Lowe MJ 1998). Comparing the NE-Tinnitus (+) group with the NE-Tinnitus (-) group, significantly increased ALFF values were observed in the paraflocculus (PFL) and AC (Fig. 5A). However, when the NE-Tinnitus (+) group was compared with the control group, multiple brain regions, including the entorhinal area (ENT), hippocampal–amygdalar transition area (HATA), supplementary somatosensory area (SSs), striatum-like amygdalar nuclei (sAMY) and medial amygdalar nucleus (MEA), presented decreased ALFF values (Fig. 5B). The ALFF values in the AC, SSs, and primary somatosensory cortex (SSp) were significantly lower in the SS-Tinnitus (+) group than in the SS-Tinnitus (-) group (Fig. 5C). When the SS-Tinnitus (+) group was compared with the control group, increased ALFF was detected in the anterior olfactory nucleus (AON), whereas decreased ALFF was detected in the medulla (MY) (Fig. 5D). Based on the above results, we believe that SS overdose and traumatic noise exposure may induce tinnitus through different central mechanisms, leading to diverse patterns of ALFF activation. Fig.5. Significant enhancement and depression of amplitude of low-frequency fluctuation (ALFF) for noise exposure group and sodium salicylate group respectively. (A) ALFF increases in PFL and AC, NE-Tinnitus (+) group mice versus NE-Tinnitus (-) group mice, at 14th day after traumatic noise exposure. (B) ALFF decreases in ENT, HATA, SSs, sAMY and MEA, NE-Tinnitus (+) group mice versus control mice, at 14th day after traumatic noise exposure. (C) ALFF decreases in AC, SSs, and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. (D) ALFF changes in MY, pons and AON, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p<0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: PFL, paraflocculus; AC, auditory cortex; ENT, entorhinal area; HATA, hippo-campo amygdalar transition area; SSs, supplementary somatosensory area; sAMY, striatum-like amygdalar nuclei; MEA, medial amygdalar nucleus; CA1, Ammon’s horn; AI, agranular insular area; STR, striatum; PAL, pallidum; MOB, main olfactory bulb; MY, medulla; AON, anterior olfactory nucleus; SSp, primary somatosensory cortex. The alterations of regional homogeneity (ReHo) in overdose sodium salicylate and traumatic noise exposure group A As a voxel-based measure of brain activity, ReHo is used to evaluate the similarity or synchronization between the time series of a given voxel and its nearest neighbors (Zang et al. 2004), ased on the hypothesis that intrinsic brain activity is manifested by clusters of voxels rather than single voxels. Higher ReHo indicates that neurons in that specific region tend to synchronize and that there is increased consistency of neuronal activity. On the other hand, low ReHo indicates decreased consistency of neuronal activity, which may suggest dysfunction in the brain region (Qu T 2019). In the NE-Tinnitus (+) group, the AC, Ammon's horn (CA) and striatum (STR) had significantly greater ReHo values than those in the control group (Fig. 6A) . However, other brain regions in the NE-Tinnitus (+) group, including the sAMY, HATA, subiculum (SUB), and midbrain (MB), presented decreased ReHo, suggesting a two-way effect of noise exposure. In the NE-Tinnitus (-) group, only decreased ReHo was observed in the HATA, ENT and cortical subplate (CTXsp) regions ( Fig. 6B ). In the SS overdose group, the ReHo exhibited a different pattern of change than that in the traumatic noise exposure group. As shown in Fig. 6C , the SS-Tinnitus (+) group presented lower ReHo values than did the control group in the CA1, AC, SSs, SSp, and retrosplenial area and lateral agranular part (RSPagl). The SS-Tinnitus (-) group presented increased ReHo values in the SSp and CA1 and decreased ReHo values in the hypothalamus (HY) and pons ( Fig. 6D ), suggesting that SS administration has an impact on ReHo, particularly in the limbic system, although it fails to induce tinnitus. When the SS-Tinnitus (+) group was compared with the SS-Tinnitus (-) group, multiple regions, including the MY, SSp, SSs, thalamus, sensory‒motor cortex-related (DORsm), hippocampus (HIP), thalamus and polymodal association cortex-related (DORpm) regions, all presented decreased ReHo in tinnitus mice ( Fig. 6E ). The ALFF and ReHo results in tinnitus mice indicated that different patterns of neural activity developed not only in auditory pathways (such as the AC) but also in limbic systems (such as the hippocampus and thalamus) and somatosensory systems (such as the SSp and SSs). On the other hand, the different brain activity patterns in the SS overdose and traumatic noise exposure groups provide evidence that these two modeling methods may induce tinnitus through different neural circuits or physiological mechanisms. Fig.6. Significant increase and decrease of regional homogeneity (ReHo) for overdose sodium salicylate and traumatic noise exposure group respectively. (A) ReHo changes in sAMY, HATA, SUB, MB, AC, CA3, and STR, NE-Tinnitus (+) group versus control, at 14th day after traumatic noise exposure. (B) ReHo decreases in ENT, HATA and CTXsp, NE-Tinnitus (-) group mice versus control mice, at 14th day after traumatic noise exposure. (C) ReHo increases in RSPagl, CA1, AC, SSs and SSp, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (D) ReHo changes in HY, pons, SSp and CA1, SS-Tinnitus (-) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (E) ReHo decreases in MY, SSp, DORsm ,SS, HIP, DORpm and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p<0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: SUB, subiculum; MB, midbrain; CTXsp, cortical subplate; RSPagl, retrosplenial area, lateral agranular; HY, hypothalamus; DORsm, thalamus, sensory-motor cortex related; HIP, hippocampus; DORpm, thalamus, polymodal association cortex related. Alterations in FC patterns in tinnitus mice induced by SS overdose and traumatic noise exposure FC refers to the similarity between signals arising from two brain regions (Mohanty et al. 2020). Higher FC may indicate that the regions tend to be functionally connected (Greicius et al. 2003; Eickhoff and Müller 2015). To investigate whether there were FC changes in tinnitus mice, 7 main brain regions in the auditory pathway were selected as ROIs, and the connectivity between any two regions was examined. In both the traumatic noise exposure group and the SS overdose group, we calculated the FC from 7 brain regions of the central auditory pathway to the AC. In the traumatic noise exposure group, most brain regions, especially the MGB region, presented increased FC in tinnitus mice ( Fig. 7A and 7C ). Interestingly, in the SS overdose group, the FC values of the brain regions in the tinnitus group and the control group were not significantly different ( Fig. 7B and 7C ). These noticeably different patterns further suggest that noise exposure and sodium salicylate may induce tinnitus through different mechanisms or neural pathways. Fig. 7. FC is calculated between the defined AC and related brain regions of interest. ( A ) Pattern diagram of related brain regions in correlation with auditory cortex. (B) FC between NE-Tinnitus (+) group (n=4) and control group (n=10). DCO (NT=0.04±0.03，Control=0.11±0.03), IC (NT=0.08±0.07，Control=0.03±0.03),MG (NT=0.09±0.06，Control=0.08±0.03), NLL (NT=-0.16±0.04，Control=0.04±0.02), SOC (NT=-0.01±0.03，Control=0.05±0.03), VOC (NT=0.03±0.03，Control=-0.002±0.02). (C) FC between SS-Tinnitus (+) group (n=6) and control group (n=10). DCO (SS=-0.01±0.06，Control=0.11±0.03), IC (NT=0.09±0.11，Control=0.03±0.03),MG (NT=0.30±0.09，Control=0.08±0.03), NLL (NT=-0.16±0.04，Control=0.04±0.02), SOC (NT=-0.01±0.03，Control=0.05±0.03), VOC (NT=0.03±0.03，Control=-0.002±0.02). (D) Comparison of FC among NE-Tinnitus (+) group, SS-Tinnitus (+) group and control group. Mean ± SEM was shown. :Level of significance is illustrated in the figures with symbols or shaded areas (not significant (n.s.): p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.001). Abbreviations: AC, auditory cortex; DCO, dorsal cochlear nucleus; IC, inferior colliculus; MG, medial geniculate body; NLL, nucleus of the lateral lemniscus; SOC, superior olivary body; VOC, ventral cochlear nucleus. Discussion Our study systematically demonstrated that mice with tinnitus induced by different methods presented different patterns in hearing threshold shift, behavioral tests and rs-fMRI physiological indices. This study creatively utilized two different methods to induce tinnitus: SS overdose and traumatic noise exposure. In our study, animals exposed to tinnitus-inducing procedures were measured for tinnitus by evaluating how silence modulated their reflexive behavioral responses evoked by unexpected suprathreshold sounds (PPIAS and GPIAS). Many animal studies using PPIAS and GPIAS have used noise exposure levels that induced TTS rather than PTS, similar to our research in which functional hearing was preserved. According to our results, SS overdose could cause a permanent ABR threshold elevation of approximately 15 dB SPL at most frequencies, whereas the temporary hearing threshold shift caused by noise exposure returned to normal after 2 weeks. Behavioral tests indicated that both methods successfully induced tinnitus in mice. Mice with tinnitus induced by different methods exhibited different proportions and characteristics, which indicates differences in the underlying mechanisms of tinnitus induction. rs-fMRI data analysis revealed significant increases in the ALFF of the PFL and AC in mice with tinnitus caused by traumatic noise exposure. In the SS overdose group, the ALFF increased in the AON but decreased in the MY and pons. ReHo analysis revealed that neurons in the AC tended to discharge in a more synchronized way in the traumatic noise exposure group, but other brain regions, such as the sAMY, HATA and MGB, presented reduced ReHo values. The tinnitus mice in the SS overdose group also presented increased ReHo values in the AC, as well as in the SSp, SSs, CA1 and RSPagl. This study also calculated the FC between brain regions by exacting time series of fMRI signals in ROIs to identify the sequence and relevance of activated brain regions. Interestingly, in the traumatic noise exposure group, tinnitus mice had significantly lower FC to the NL than did the control group. Tinnitus mice in the SS group presented significantly greater FC to the MGB than did those in the control group. Based on ALFF, ReHo, and FC analyses, the two methods may induce tinnitus in different ways. Tinnitus induced by traumatic noise exposure Noise-related factors are responsible for a significant percentage of tinnitus cases. In nearly 58% of cases, individuals subjected to acute acoustic trauma develop symptoms of tinnitus as a result of the immediate damage to the inner ear (Bhatt et al. 2016). It is widely accepted that noise exposure affects neural plasticity in the central nervous system. Increasing evidence suggests that chronic tinnitus can result from a failure of limbic structures to block hyperactive auditory signals induced by noise exposure (Rauschecker et al. 2010; Zhang et al. 2021). Noise-induced tinnitus is prevalent in humans and animals with normal audiograms because of increased neuronal activity caused by noise exposure and plasticity (Nagaraj et al. 2020; Qu et al. 2019). In individuals claiming compensation for work-related hearing loss, the prevalence of tinnitus was approximately constant over a wide hearing range (Lewkowski et al. 2023). Some authors have suggested that noise may cause tinnitus, and even tinnitus patients with normal audiograms may have limited cochlear damage. There is also evidence that not all patients with hearing loss develop tinnitus(Alshabory et al. 2022; Pavlidis et al. 2023). Our study used a level of traumatic noise exposure that induced TTS rather than PTS, similar to previous studies in mice and guinea pigs in which functional hearing was preserved (Longenecker and Galazyuk 2016; Park et al. 2020). Many studies suggest that the differences in peripheral damage and central plasticity among individual animals are due to the heterogeneity of tinnitus pathology (Hickox and Liberman 2014; Knipper et al. 2015). Tinnitus induced by SS overdose SS inhibits cyclooxygenase activity, which could disrupt the conversion of arachidonic acid into prostaglandin H2. Excessive SS could increase the probability of channel opening in the NMDA receptor (Yamakura and Shimoji 1999). SS can cause tinnitus by overactivating NMDA receptors, resulting in neuronal hyperexcitation and tonotopic shifts in auditory pathways (Ruel et al. 2008; Jiang et al. 2017). Extracellular recordings in vitro have indicated that salicylate can drastically alter the spontaneous firing rate (SFR) of neurons in the MGB, although the direction of change is complex. Approximately 52.4% of neurons increased their firing rate after SS treatment, whereas firing rates decreased in approximately 47.6% of neurons (Basta et al. 2008; Ma et al. 2006). Since the MGB provides excitatory inputs to the primary AC (A1), changes in the MGB are likely to significantly impact activity in A1. The preceding results demonstrated that salicylate not only suppresses the neural output of the peripheral auditory system but also alters activity in the hCNS (Stolzberg et al. 2011). I In some cases, SFRs in A1 and the anterior auditory field (AAF) decreased slightly after salicylate treatment, whereas those in the secondary AC (A2) increased. A1 neurons receive afferent inputs mainly from the lemniscal pathway, and A2 neurons receive afferent information from the extralemniscal pathway (Eggermont and Kenmochi 1998). The reduction in the A1 SFR after SS treatment may therefore be due primarily to the suppression of neural output from the cochlea and classical auditory pathway, whereas the enhanced spontaneous firing observed in A2 may reflect the changes occurring at both auditory and non-auditory loci in the CNS (Huang and Winer 2000). PPIAS and GPIAS Unlike humans, in whom tinnitus can be self-reported, animal models face exceptional challenges in reliably inducing and detecting tinnitus. There are also complex variations in the perception of tinnitus depending on background noise, stress, fatigue, and other factors (Clifford et al. 2019). PPIAS and GPIAS are reliable methods for the detection of tinnitus. In the salicylate tinnitus model, GPIAS typically decreases in the low-frequency range compared with the range between 8 and 16 kHz (Berger et al. 2013). The GPIAS deficiency in the salicylate model usually returns to normal within 72 hours after the last administration, so we conducted the appropriate tests within 24 hours after the last administration of SS in our study (Stolzberg et al. 2012). However, the prerequisite for normal hearing limits the usage opportunities for GPIAS. Hearing loss caused by noise exposure or ototoxic drugs can make background sound and the embedded gaps less audible(Fournier and Hébert 2013). Therefore, when hearing loss is induced in one ear to trigger tinnitus, the other ear should be protected. To ensure the accuracy of PPIAS and GPIAS, we protected the right ear of the mice in the traumatic noise exposure group using an earplug. AC and tinnitus Salicylate-induced tinnitus leads to reduced SFRs in the AC (Noreña et al. 2010). Our results also revealed decreased synchronization and neuronal density with spontaneous synchronized firing in the AC induced by SS overdose. However, a significant increase in metabolic activity in the AC and inferior colliculus was detected via micropositron emission tomography in an animal model, potentially reflecting increased inhibitory synaptic activity (Paul et al. 2009). The reduction in SFR in the AC, as well as the increase in the SFRs of the second auditory cortical field and external nucleus of the IC, suggest that while the AC is involved in hyperacusis-like effects, the extralemniscal pathway is the primary cause of salicylate-induced tinnitus(Manabe et al. 1997). Our findings also strongly suggested that traumatic noise exposure could cause increased cortical reorganization, SFRs and interneuronal synchrony in the AC, which is similar to the findings of previous studies (Eggermont 2015). As determined behaviorally by conditioned response testing, the AC could exhibit increased SFRs as a result of downregulation of inhibition in the tinnitus region according to Yang et al.’s research in a traumatic noise animal model (Yang et al. 2011). MGB and tinnitus The MGB is a mandatory relay station along the auditory pathway that mediates the thalamocortical network involved in tinnitus (Chen et al. 2015). Classical and non-classical ascending auditory pathways contribute differently to auditory stimuli processing in the MGB and are most likely to contribute to tinnitus pathophysiology (Pickles 2015). The evidence across several species indicates that the MGB dynamically shapes simple tones and complex vocalizations before auditory sensations reach the cerebral cortex (Mihai et al. 2019; Kraus et al. 1994; Cai et al. 2016). Previous studies have shown that animals with tinnitus have an increased number of spikes per burst and tonic GABAA currents, which leads to increased output from the MGB to higher auditory cortices (Sametsky et al. 2015). In a tinnitus model induced by SS, when assessing the coherence between the MGB and PAC, SS enhanced coherence in the gamma band, which was suggested to be a direct neural correlate of tinnitus, influencing thalamocortical networks (Vianney-Rodrigues et al. 2019). However, spontaneous firing rates in the MGB have been found to be unaffected in rats with tinnitus induced by acoustic noise trauma (Barry et al. 2019). Like in previous studies, MGB excitability was observed only in mice with SS overdose-induced tinnitus and not in mice with traumatic noise exposure-induced tinnitus in our study (Brinkmann et al. 2021). According to neuroimaging studies on tinnitus patients, there is decreased FC between the thalamus and superior frontal gyrus and increased spontaneous neural activity between the two areas (Zhang et al. 2015; Chen et al. 2014). The contribution of the MGB to tinnitus can possibly be explained by the fact that unpleasant auditory inputs are normally "cancelled out" at the level of the MGB. However, the noise cancellation mechanism in patients with tinnitus is dysfunctional, resulting in disinhibition of the MGB, which may contribute to the perception of tinnitus sounds (Rauschecker et al. 2010; Elgoyhen et al. 2015). Conclusion Overall, this study aimed to investigate the neural basis of tinnitus by comparing two commonly used animal models: traumatic noise exposure and SS overdose. Both methods were found to successfully induce tinnitus in mice, as evidenced by behavioral tests. However, rs-fMRI analysis revealed distinct patterns of neural activity in tinnitus mice from each group, suggesting differences in the underlying mechanisms of tinnitus induction. These findings suggest that traumatic noise exposure and SS overdose induce tinnitus through distinct neural mechanisms involving different neural circuits and pathways. Understanding these differences is crucial for developing targeted therapies for tinnitus. Declarations Funding: This work was supported by the Natural Science Foundation of Shanghai No 19ZR1408700, Shanghai 2020 "Science and Technology Innovation Action Plan"- "One Belt One Road" International Cooperation Project (20410740600), Clinical Research Plan of SHDC (SHDC2020CR1049B), General Project of Shanghai Hongkou District Health Commission（2102-21）and the Key Clinical Specialty Construction Project of Shanghai Hongkou District (HKLCZD2024A03). Competing interests: The authors declare no competing interests. Author Contributions: conceptualisation: W.Z., J.N. data curation: W.Z., J.N, Y.Z, R.M. software: W.Z., J.N. formal analysis: W.Z., J.N, Y.Z, R.M. visualisation: W.Z., J.N., J.Y. methodology: M.C. writing (original draft): W.Z., J.S. supervision: M.C., J.Y. funding acquisition: W.Z., J.S., M.C., J.Y. writing (review and editing): M.C., J.Y. Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval: This study was approved by the Ethics Committee of the Eye Ear Nose & Throat Hospital of Fudan University (No.2021-2021220). All the experimental animals were treated in accordance with the guidelines of the Ethical Board of EENT Hospital, Fudan University. References Adjamian P, Hall DA, Palmer AR, Allan TW, Langers DRM (2014) Neuroanatomical abnormalities in chronic tinnitus in the human brain. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5014033","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":361118706,"identity":"6d4063c1-df96-4604-81c1-986589e50a0f","order_by":0,"name":"Weixun Zhang","email":"","orcid":"","institution":"Eye \u0026 ENT Hospital of Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Weixun","middleName":"","lastName":"Zhang","suffix":""},{"id":361118707,"identity":"9d3cc329-f729-48f5-80b0-7d2e28a63b8e","order_by":1,"name":"Jianan Shi","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jianan","middleName":"","lastName":"Shi","suffix":""},{"id":361118708,"identity":"35fd329b-ad82-4785-a9ac-4103a169790c","order_by":2,"name":"Yuwen Zhang","email":"","orcid":"","institution":"Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuwen","middleName":"","lastName":"Zhang","suffix":""},{"id":361118709,"identity":"1b1560eb-9059-4631-9bec-72b8039e287c","order_by":3,"name":"Rui Ma","email":"","orcid":"","institution":"Eye \u0026 ENT Hospital of Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Ma","suffix":""},{"id":361118710,"identity":"9d41f065-b77e-42db-a15c-15c40d8c3b54","order_by":4,"name":"Meirong Chen","email":"","orcid":"","institution":"Jiangwan Hospital of Hongkou District","correspondingAuthor":false,"prefix":"","firstName":"Meirong","middleName":"","lastName":"Chen","suffix":""},{"id":361118711,"identity":"8ea82cc7-58ac-47ef-9781-ff559cda82f3","order_by":5,"name":"Juanmei Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYLACCTDJ2HDgQ4WNHBt78wGitTQenHEmzZiP51gC0ZYxH+ZtO5w4TyJHAa8yg+NnD7+wbLPL021vbjjMc4Y5vY0hh4HhR8U23FrO5KVZSLYlF5udOdhwcE4FW24bw9kDjD1nbuPWciDHzECyjTlx243EhgNvzvDktjH2JTAztuHRcv4NSEt94rb7DxsO8LZJpLMx8xjg13Ijx/iBJNDX224wNhzkbTNIYGMjoEXyxhszBolzxxO3nUlsAAZygmEbD1vCQXx+4TufY/xZoqw6cdvx448/fKj4Ly8///HBBz8qcGtROMDAJi2BLnoAp3ogkG9gYP74AZ+KUTAKRsEoGAUA/bRlcA9WM1oAAAAASUVORK5CYII=","orcid":"","institution":"Eye \u0026 ENT Hospital of Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Juanmei","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-09-01 17:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5014033/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5014033/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67196616,"identity":"56abbaf9-934e-4f9d-8617-acb7bb969e09","added_by":"auto","created_at":"2024-10-22 09:12:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62438,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental timeline. The animals were randomly assigned to three groups: the control group, the traumatic noise exposure group and the overdose sodium salicylate group.\u003cstrong\u003e \u003c/strong\u003eBaseline hearing levels (Day 0) were measured through ABRs in all groups. \u003cstrong\u003e(A) \u003c/strong\u003eThe animals in the traumatic noise exposure group received 100dB SPL 8-16kHz narrowband noise exposure for 2 hours. ABRs evaluations were preformed in 1 day, 7 days, and 14 days after the traumatic noise exposure. Behavioral tests and rs-fMRI scan were only conducted 14 days after noise exposure. \u003cstrong\u003e(B) \u003c/strong\u003eIn the overdose sodium salicylate group, the mice were given 350mg/kg sodium salicylate intraperitoneally once daily for 7 successive days. The mice were conducted ABRs, behavioral tests and rs-fMRI scan in 1 day after the end time point of sodium salicylate injection.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/3c41577ddc14b71f89b378f0.png"},{"id":67196612,"identity":"52eb7dab-67be-48d9-847d-6ec631b1fb66","added_by":"auto","created_at":"2024-10-22 09:12:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145496,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Sodium salicylate injection produce long-term, significant hearing threshold shift. On day 7 of continuous injection, hearing thresholds at each frequency are 74.375±1.475 dB SPL (4kHz, P=0.1234), 45.000±1.637 dB SPL (8kHz, P=0.0019), 41.250±1.250 dB SPL (16kHz, P\u0026lt;0.0001), 46.875±1.315 dB SPL (24kHz, P\u0026lt;0.0001), and 55.625±1.990 dB SPL (32kHz, P\u0026lt;0.0001). (B) Noise exposure produce permanent, reversible hearing impairment. On day 1 right after noise exposure, hearing threshold was significantly elevated at all frequencies but recovered to normal on day 14 after noise exposure. A maximum threshold shift of over 30 dB was detected at 32 kHz at day 1 post-exposure and recovered to normal pre-exposure values by day 14. Mean ± SEM is shown in each group. Paired t-test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/d88ba74c0a868f916169c639.png"},{"id":67196610,"identity":"3bb5ab12-e6a9-49e5-8b9a-ce7b0104fad8","added_by":"auto","created_at":"2024-10-22 09:12:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234995,"visible":true,"origin":"","legend":"\u003cp\u003e∆PPIAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆PPIAS ratio were calculated on BBN, 8kHz, 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. \u003cstrong\u003e(A-E)\u003c/strong\u003e ∆PPIAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). \u003cstrong\u003e(F) \u003c/strong\u003eComparison of average ∆PPIAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean ± SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group.p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p\u0026lt; 0.001 by two-way ANOVA. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p\u0026lt; 0.001 by two-way ANOVA. The black hashes represent the statistical difference between the NE-Tinnitus (+) and the NE-Tinnitus (-) group. p \u0026gt; 0.05; #: p \u0026lt; 0.05; ##: p \u0026lt; 0.01; ###: p \u0026lt; 0.001; ####: p\u0026lt; 0.001 by two-way ANOVA.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/75cf0246dba7af334692f33a.png"},{"id":67196614,"identity":"d0976566-567a-4f85-a7ec-ba8dc2854c73","added_by":"auto","created_at":"2024-10-22 09:12:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215843,"visible":true,"origin":"","legend":"\u003cp\u003e∆GAPAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆GAPAS ratio were calculated on 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. \u003cstrong\u003e(A-E)\u003c/strong\u003e ∆GAPAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). \u003cstrong\u003e(F) \u003c/strong\u003eComparison of average ∆GAPAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean ± SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group.p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p\u0026lt; 0.001 by two-way ANOVA. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p\u0026lt; 0.001 by two-way ANOVA. The black hashes represent the statistical difference between the NE-Tinnitus (+) and the NE-Tinnitus (-) group. p \u0026gt; 0.05; #: p \u0026lt; 0.05; ##: p \u0026lt; 0.01; ###: p \u0026lt; 0.001; ####: p\u0026lt; 0.001 by two-way ANOVA.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/efd5c83a7ca3a93dd5c16c75.png"},{"id":67197365,"identity":"8336fa26-6188-45f4-82f3-6da3a53156c0","added_by":"auto","created_at":"2024-10-22 09:20:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":643242,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant enhancement and depression of amplitude of low-frequency fluctuation (ALFF) for noise exposure group and sodium salicylate group respectively. \u003cstrong\u003e(A) \u003c/strong\u003eALFF increases in PFL and AC, NE-Tinnitus (+) group mice versus NE-Tinnitus (-) group mice, at 14th day after traumatic noise exposure.\u003cstrong\u003e (B) \u003c/strong\u003eALFF decreases in ENT, HATA, SSs, sAMY and MEA, NE-Tinnitus (+) group mice versus control mice, at 14th day after traumatic noise exposure.\u003cstrong\u003e (C) \u003c/strong\u003eALFF decreases in AC, SSs, and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. \u003cstrong\u003e(D)\u003c/strong\u003e ALFF changes in MY, pons and AON, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p\u0026lt;0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: PFL, paraflocculus; AC, auditory cortex; ENT, entorhinal area; HATA, hippo-campo amygdalar transition area; SSs, supplementary somatosensory area; sAMY, striatum-like amygdalar nuclei; MEA, medial amygdalar nucleus; CA1, Ammon’s horn; AI, agranular insular area; STR, striatum; PAL, pallidum; MOB, main olfactory bulb; MY, medulla; AON, anterior olfactory nucleus; SSp, primary somatosensory cortex.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/8bbb4f7405a5bbf4f69ce7f2.png"},{"id":67197366,"identity":"a5298727-d714-4536-b3f6-626b53498d22","added_by":"auto","created_at":"2024-10-22 09:20:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":856458,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant increase and decrease of regional homogeneity (ReHo) for overdose sodium salicylate and traumatic noise exposure group respectively. (A) ReHo changes in sAMY, HATA, SUB, MB, AC, CA3, and STR, NE-Tinnitus (+) group versus control, at 14th day after traumatic noise exposure. (B) ReHo decreases in ENT, HATA and CTXsp, NE-Tinnitus (-) group mice versus control mice, at 14th day after traumatic noise exposure. (C) ReHo increases in RSPagl, CA1, AC, SSs and SSp, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (D) ReHo changes in HY, pons, SSp and CA1, SS-Tinnitus (-) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (E) ReHo decreases in MY, SSp, DORsm ,SS, HIP, DORpm and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p\u0026lt;0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: SUB, subiculum; MB, midbrain; CTXsp, cortical subplate; RSPagl, retrosplenial area, lateral agranular; HY, hypothalamus; DORsm, thalamus, sensory-motor cortex related; HIP, hippocampus; DORpm, thalamus, polymodal association cortex related.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/9097c4cad03c6c1a343f1402.png"},{"id":67196617,"identity":"8c489451-8d34-4530-ac50-63950a37d5a0","added_by":"auto","created_at":"2024-10-22 09:12:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":169678,"visible":true,"origin":"","legend":"\u003cp\u003eFC is calculated between the defined AC and related brain regions of interest. (\u003cstrong\u003eA\u003c/strong\u003e) Pattern diagram of related brain regions in correlation with auditory cortex. \u003cstrong\u003e(B)\u003c/strong\u003eFC between NE-Tinnitus (+) group (n=4) and control group (n=10). DCO (NT=0.04±0.03，Control=0.11±0.03), IC (NT=0.08±0.07，Control=0.03±0.03),MG (NT=0.09±0.06，Control=0.08±0.03), NLL (NT=-0.16±0.04，Control=0.04±0.02), SOC (NT=-0.01±0.03，Control=0.05±0.03), VOC (NT=0.03±0.03，Control=-0.002±0.02).\u003cstrong\u003e (C) \u003c/strong\u003eFC between SS-Tinnitus (+) group (n=6) and control group (n=10). DCO (SS=-0.01±0.06，Control=0.11±0.03), IC (NT=0.09±0.11，Control=0.03±0.03),MG (NT=0.30±0.09，Control=0.08±0.03), NLL (NT=-0.16±0.04，Control=0.04±0.02), SOC (NT=-0.01±0.03，Control=0.05±0.03), VOC (NT=0.03±0.03，Control=-0.002±0.02).\u003cstrong\u003e(D) \u003c/strong\u003eComparison of FC among NE-Tinnitus (+) group, SS-Tinnitus (+) group and control group. Mean ± SEM was shown. :Level of significance is illustrated in the figures with symbols or shaded areas (not significant (n.s.): p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001). Abbreviations: AC, auditory cortex; DCO, dorsal cochlear nucleus; IC, inferior colliculus; MG, medial geniculate body; NLL, nucleus of the lateral lemniscus; SOC, superior olivary body; VOC, ventral cochlear nucleus.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/dedf0047802959e1bc38c771.png"},{"id":79375900,"identity":"2740984b-e67d-4df9-97b3-8b2d0f412e42","added_by":"auto","created_at":"2025-03-27 15:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3329195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5014033/v1/deb8e40d-3886-4b32-a00c-f9bb2ab92a31.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Resting-state Functional MRI Study of Mice with Tinnitus Induced by Traumatic Noise Exposure and Sodium Salicylate Overdose","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTinnitus, a phantom auditory sensation that dramatically and negatively affects an individual\u0026rsquo;s quality of life and social interactions, affects approximately 10\u0026ndash;15% of the general population. Severe tinnitus affects approximately 1\u0026ndash;2% of the global population\u0026nbsp;(Langguth et al. 2013; Auerbach et al. 2014). Unfortunately, there is currently no widely effective therapy that eliminates tinnitus, perhaps because its neural basis remains elusive.\u003c/p\u003e\n\u003cp\u003eMost cases of chronic tinnitus are associated with central gain changes, including neuronal hyperactivity, bursting discharges and increased cortical neural synchrony in the auditory system, which occurs after deafferentation of auditory nerves\u0026nbsp;(Adjamian et al. 2014; Turrigiano 2012). Predictive brain frameworks suggest that changes in cortical homeostatic plasticity in response to central gain changes are part of the pathogenesis of tinnitus \u0026nbsp;(Nore\u0026ntilde;a and Farley 2013). ; the role of these changes in tinnitus pathogenesis is still unknown but may result from\u0026nbsp;a lack of inhibition in the central auditory pathway.\u003c/p\u003e\n\u003cp\u003eT The use of animal models to study tinnitus is highly valuable, as these models allow for strict experimental control that is not permissible in human research\u0026nbsp;(Henton and Tzounopoulos 2021). The systemic application of an overdose of sodium salicylate (SS) and traumatic noise exposure are two commonly used methods for inducing tinnitus in animals. Although both salicylate and traumatic noise can produce tinnitus symptoms that are similar to those of chronic tinnitus patients, the underlying mechanisms are likely different.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe blood oxygen level-dependent (BOLD) signal observed on magnetic resonance imaging (MRI) measures neuronal activity using the ratio of oxyhemoglobin to deoxyhemoglobin as a contrast mechanism\u0026nbsp;(Leuthardt et al. 2015). The accumulation of spontaneous, highly synchronized, short time-lag neural activations between two groups of neurons in two separate regions is reflected in a positively correlated, homogeneous hemodynamic BOLD signal obtained during resting-state functional MRI (fMRI)(Cardin et al. 2023). A functional association network of executive functions and cognitive control has been successfully constructed using resting-state functional connectivity (FC)\u0026nbsp;(Cohen and D\u0026apos;Esposito 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe goal of our study was to map the regions of neural hyperactivity and enhanced FC that characterize the central gain changes in mice with tinnitus induced by SS overdose and traumatic noise exposure. Behavioral task performance was used to identify the characteristic tinnitus frequencies in the mice. We subsequently used rs-fMRI to explore potential changes in the fractional amplitude of low-frequency fluctuations (fALFF) and FC of the core brain regions in the auditory pathway. Our results demonstrated that the medial geniculate body (MGB) and auditory cortex (AC) had the highest degree of hypersensitivity in the tinnitus mouse model, indicating the potential vital role of the MGB in the pathology of tinnitus.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult CBA/Ca mice (n=37) weighing ~25 g and aged ~6 weeks at the start of the experiments were used in this study. The mice were housed 3\u0026ndash;5 animals per cage within a colony room with a 12-h light‒5-h dark cycle at 23\u0026deg;C. All animal procedures were approved by the Ethics Committee of the Eye Ear Nose \u0026amp; Throat Hospital of Fudan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTraumatic noise exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice assigned to the noise exposure group were placed in custom-made metal cages before being moved to soundproof boxes containing four speakers for noise exposure. The difference between the noise sound pressure level received by the mouse\u0026apos;s ears and the target sound pressure level (SPL) was less than 1 dB. Noise was generated by a TDT System 3 (TDT, USA) , amplified through a P9500S Power Amplifier (YAMAHA, Japan), and played through 4 Pyramid Tweeter TW67 speakers (Pyramid, USA). The parameters of the noise exposure protocol were 8\u0026ndash;16 kHz narrowband noise at an intensity of 100 dB SPL for 2 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverdose sodium salicylate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSS (Sigma‒Aldrich, USA) was administered\u0026nbsp;to\u0026nbsp;mice in\u0026nbsp;the ototoxic drug overdose group through intraperitoneal (i.p.) injection. Previous studies have\u0026nbsp;shown\u0026nbsp;that even through systemic administration, SS can pass through the\u0026nbsp;blood\u0026ndash;brain\u0026nbsp;barrier,\u0026nbsp;increasing\u0026nbsp;the SS concentration in cerebrospinal fluid [11, 12]. Studies have also shown that\u0026nbsp;250~400 mg/kg is an\u0026nbsp;effective concentration depending on\u0026nbsp;the\u0026nbsp;animal species [13]. In this\u0026nbsp;study, we chose\u0026nbsp;350 mg/kg i.p. injection for 1 week to induce tinnitus. Injections were performed at approximately the same time\u0026nbsp;every day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuditory brainstem responses (ABRs) test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hearing thresholds of animals\u0026nbsp;exposed to\u0026nbsp;noise and ototoxic drugs were measured,\u0026nbsp;and the ABRs were recorded.\u0026nbsp;The animals\u0026nbsp;were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and placed in a soundproof box (Tucker-Davis Technologies, USA) before\u0026nbsp;testing. Five frequencies (4, 8, 16, 24 and 32 kHz) were tested using a small speaker placed 10 cm from\u0026nbsp;the midpoint\u0026nbsp;of the two ears (MF1\u0026nbsp;multifield\u0026nbsp;magnetic speakers and P9500S YAMAHA power amplifier), and the results were recorded.\u0026nbsp;The waveforms\u0026nbsp;were visualized and recorded using BioSigRZ software and\u0026nbsp;a TDT System 3 data acquisition system (Tucker-Davis Technologies, USA). The sound stimulus started at a 90 dB SPL and was decreased\u0026nbsp;by\u0026nbsp;5 dB until the hearing threshold was determined. For each SPL, 600 responses were recorded and averaged.\u0026nbsp;Offline analysis of stored waveforms was used to determine ABR wave 1 thresholds. The hearing threshold was defined as the lowest stimulus level that elicited repeatable wave 1 responses on visual inspection of stacked waveforms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral tests of tinnitus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrepulse\u0026nbsp;inhibition of the acoustic startle reflex (PPIAS) and\u0026nbsp;gap\u0026nbsp;prepulse inhibition of the acoustic startle reflex (GPIAS) were tested in all\u0026nbsp;the mice before tinnitus induction and 7 d after SS injection or 14 d after noise exposure using a custom-built system. All behavioral tests were conducted in a sound-attenuating cubicle.\u0026nbsp;The sounds\u0026nbsp;were presented through\u0026nbsp;a standard acoustic startle sound speaker (MED Associates, USA), which was positioned in front of\u0026nbsp;the animals. A narrow bandpass-filtered background sound (1 kHz\u0026nbsp;bandwidth centered at 10, 16, 20, 24 and 32 kHz) was presented at\u0026nbsp;60 dB\u0026nbsp;SPL for\u0026nbsp;17~25 s\u0026nbsp;(randomly varied) before\u0026nbsp;the presentation of an acoustic startle stimulus (white noise,\u0026nbsp;115 dB\u0026nbsp;SPL,\u0026nbsp;20 ms).\u0026nbsp;The entire\u0026nbsp;session consisted of 15 startle-only trials, 15 broadband noise (BBN) trials and 10 gap detection trials for each frequency. Gap detection trials were identical to startle-only trials except that a 50-ms silent gap was inserted\u0026nbsp;130 ms\u0026nbsp;before the startle stimulus. For each frequency, 10 gap/no-gap ratios were collected and averaged, and\u0026nbsp;the gap detection ability was determined using the gap detection ratio, calculated as the ratio of\u0026nbsp;the startle response waveform peak value with gaps\u0026nbsp;to the\u0026nbsp;peak value without gaps. For a given frequency,\u0026nbsp;a gap startle ratio closer to 0\u0026nbsp;indicated\u0026nbsp;a greater GPIAS,\u0026nbsp;and\u0026nbsp;a higher gap startle ratio\u0026nbsp;indicated a\u0026nbsp;defect in gap detection. The PPIAS is essentially the inverse of the GPIAS, except that the 50-ms silent gaps in the GPIAS were replaced with a\u0026nbsp;70 dB\u0026nbsp;SPL,\u0026nbsp;50 ms\u0026nbsp;prepulse with different frequencies (8, 10, 16, 20, 24,\u0026nbsp;32 kHz\u0026nbsp;and BBN). The calculation for\u0026nbsp;the PPIAS prepulse detection ratio is identical to that of\u0026nbsp;the GPIAS gap detection ratio; frequencies with a\u0026nbsp;∆ratio of \u0026ge;0.2 were called\u0026nbsp;tinnitus frequencies. Mice\u0026nbsp;with the tinnitus frequency were considered tinnitus mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResting state functional magnetic resonance imaging (rs-fMRI) scanning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMRI scanning was carried out\u0026nbsp;at the\u0026nbsp;Zhangjiang Brain Imaging Centre.\u0026nbsp;Each\u0026nbsp;mouse was placed in\u0026nbsp;an\u0026nbsp;anesthesia box with isoflurane before\u0026nbsp;the test (induction concentration,\u0026nbsp;5%;\u0026nbsp;maintenance concentration,\u0026nbsp;0.5%) (Zoetic, USA). To simulate the blood flow of awake animals, dexmedetomidine (Zoetic, USA) was administered by i.p. injection into each mouse (0.03 mg/kg for embolism, 0.015 mg/kg for continuous subcutaneous injection). During the image acquisition procedures, the respiratory rate of\u0026nbsp;each mouse was maintained between\u0026nbsp;40\u0026ndash;60\u0026nbsp;breaths/min; the rectal temperature was maintained at\u0026nbsp;36\u0026deg;C\u0026ndash;37\u0026deg;C; the oxygen saturation was maintained at\u0026nbsp;98\u0026ndash;100%;\u0026nbsp;and the heart rate was maintained between\u0026nbsp;200\u0026ndash;300\u0026nbsp;beats/min.\u003c/p\u003e\n\u003cp\u003eMRI data were\u0026nbsp;obtained\u0026nbsp;on a 7.0-T animal MRI scanner (Bruker BioSpec 94/30, Ettlingen, Germany). For functional imaging, 30 axial slices were acquired using gradient-echo echo-planar imaging (GE-EPI) with the following parameters: repetition time (TR) =\u0026nbsp;2 s, echo time (TE) =\u0026nbsp;15 ms, matrix size = 80 \u0026times; 67, field of view (FOV) = 30 \u0026times; 30 mm\u003csup\u003e2\u003c/sup\u003e, and voxel size\u0026nbsp;=\u0026nbsp;0.375 \u0026times; 0.373 \u0026times; 0.6 mm\u003csup\u003e3\u003c/sup\u003e. High-resolution anatomical T2-weighted fast spin‒echo\u0026nbsp;images were\u0026nbsp;acquired at the same slice location with the following parameters: TR/TE = 3735 ms/33 ms, matrix = 256 \u0026times; 256, FOV = 30 \u0026times; 30 mm\u003csup\u003e2\u003c/sup\u003e, and average = 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRI data processing and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll preprocessing and data analysis were performed\u0026nbsp;with\u0026nbsp;Statistical Parametric Mapping (SPM12) software (Welcome Department of Imaging Science; http://www.fil.ion.ucl.ac.uk/spm) and DPARSF (http://rfmri.org/DPARSF) according to standard procedures, including removal of\u0026nbsp;the first 10 timepoints, slice timing, realignment, spatial normalization, smoothing, removal of the linear trend and ALFF calculation.\u003c/p\u003e\n\u003cp\u003eVoxelwise\u0026nbsp;statistical analysis\u0026nbsp;was\u0026nbsp;performed using the framework of a general linear model (GLM). To identify significant differences between groups,\u0026nbsp;a two-sample t test was carried out using SPM12, and brain regions with significant ALFF and regional homogeneity (ReHo) changes were\u0026nbsp;identified according to\u0026nbsp;a voxel-level height threshold pf p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eFor\u0026nbsp;the resting-state analysis, 7 main brain regions on\u0026nbsp;the auditory pathway were selected as regions of interest (ROIs), including\u0026nbsp;the AC, dorsal cochlear nucleus (DCN), inferior colliculus (IC), MGB, nucleus of\u0026nbsp;the lateral lemniscus (NL), superior olivary body (SOB),\u0026nbsp;and ventral cochlear nucleus (VCN).\u0026nbsp;FC\u0026nbsp;was calculated using correlation analysis among the ROIs, and time courses from the average signal in each ROI were obtained. Two-sample t tests were used to compare differences in connectivity between the experimental groups and the control group. A P value less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 7 software (Prism 7; GraphPad Software Inc., La Jolla, CA, USA). Normally distributed continuous variables are presented as the means \u0026plusmn; standard errors of the means (SEMs) or means \u0026plusmn; standard deviations (SDs). Unless otherwise noted, the level of significance is illustrated in the figures with the following symbols or shaded areas (not significant (n.s.): p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.1.\u003c/strong\u003e Experimental timeline. The animals were randomly assigned to three groups: the control group, the traumatic noise exposure group and the overdose sodium salicylate group.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBaseline hearing levels (Day 0) were measured through ABRs in all groups. \u003cstrong\u003e(A)\u0026nbsp;\u003c/strong\u003eThe animals in the traumatic noise exposure group received 100dB SPL 8-16kHz narrowband noise exposure for 2 hours. ABRs evaluations were preformed in 1 day, 7 days, and 14 days after the traumatic noise exposure. Behavioral tests and rs-fMRI scan were only conducted 14 days after noise exposure. \u003cstrong\u003e(B)\u0026nbsp;\u003c/strong\u003eIn the overdose sodium salicylate group, the mice were given 350mg/kg sodium salicylate intraperitoneally once daily for 7 successive days. The mice were conducted ABRs, behavioral tests and rs-fMRI scan in 1 day after the end time point of sodium salicylate injection.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSS injection\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eproduces a\u0026nbsp;hearing threshold shift,\u0026nbsp;and noise exposure\u0026nbsp;produces a\u0026nbsp;temporary hearing threshold shift\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eABR thresholds were analyzed to determine the\u0026nbsp;degree of\u0026nbsp;hearing loss\u0026nbsp;in\u0026nbsp;the mice in both\u0026nbsp;the\u0026nbsp;SS injection group and\u0026nbsp;the\u0026nbsp;noise exposure group. Tests were conducted on\u0026nbsp;days\u0026nbsp;0 and 7 of continuous injection for the SS group and on\u0026nbsp;days\u0026nbsp;0, 1, 7 and 14 after noise exposure for the noise exposure group.\u0026nbsp;Compared with before\u0026nbsp;injection,\u0026nbsp;7 days\u0026nbsp;after SS injection, the hearing threshold of the mice (n=10) was significantly increased at\u0026nbsp;almost all frequencies except 4 kHz (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2A\u003c/strong\u003e).\u0026nbsp;Similarly, we also found a significant threshold shift at\u0026nbsp;days\u0026nbsp;1 and 7 in\u0026nbsp;the\u0026nbsp;noise exposure group. However, on day 14 after noise exposure, the increase in hearing threshold had reversed (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2B\u003c/strong\u003e). Based on these results, we concluded that SS produced a permanent threshold shift (PTS), whereas noise exposure produced a temporary threshold shift (TTS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.2.\u003c/strong\u003e (A) Sodium salicylate injection produce long-term, significant hearing threshold shift. On day 7 of continuous injection, hearing thresholds at each frequency are 74.375\u0026plusmn;1.475 dB SPL (4kHz, P=0.1234), 45.000\u0026plusmn;1.637 dB SPL (8kHz, P=0.0019), 41.250\u0026plusmn;1.250 dB SPL (16kHz, P\u0026lt;0.0001), 46.875\u0026plusmn;1.315 dB SPL (24kHz, P\u0026lt;0.0001), and 55.625\u0026plusmn;1.990 dB SPL (32kHz, P\u0026lt;0.0001). (B) Noise exposure produce permanent, reversible hearing impairment. On day 1 right after noise exposure, hearing threshold was significantly elevated at all frequencies but recovered to normal on day 14 after noise exposure. A maximum threshold shift of over 30 dB was detected at 32 kHz at day 1 post-exposure and recovered to normal pre-exposure values by day 14. Mean \u0026plusmn; SEM is shown in each group. Paired t-test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea tinnitus mouse model by SS overdose and traumatic noise exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the effectiveness of SS overdose and traumatic noise exposure in inducing tinnitus in\u0026nbsp;a\u0026nbsp;mouse model, we conducted behavioral tests (PPIAS and GPIAS) in both experimental groups. The SS overdose group was tested 1 day after continuous injection, whereas\u0026nbsp;the traumatic noise exposure group was tested 14 days after noise exposure.\u003c/p\u003e\n\u003cp\u003eMice that exhibited the tinnitus frequency in both\u0026nbsp;the\u0026nbsp;PPIAS and GPIAS tests\u0026nbsp;were\u0026nbsp;defined as tinnitus mice in our study. According to the PPIAS prepulse detection ratio and GPIAS gap detection ratio, we divided these mice into four groups:\u0026nbsp;traumatic\u0026nbsp;noise exposure with tinnitus (NE-Tinnitus (+)) group,\u0026nbsp;traumatic\u0026nbsp;noise exposure without tinnitus (NE-Tinnitus (-)) group, SS overdose group with tinnitus (SS-Tinnitus (+)) group and SS overdose without tinnitus (SS-Tinnitus (-)) group\u003cstrong\u003e\u0026nbsp;(Figs.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3 and 4)\u003c/strong\u003e. Six mice (6/10)\u0026nbsp;in the\u0026nbsp;SS injection group\u0026nbsp;and\u0026nbsp;four (4/10) in\u0026nbsp;the\u0026nbsp;traumatic noise exposure group\u0026nbsp;presented behavioral evidence of tinnitus.\u003c/p\u003e\n\u003cp\u003eA comparison of the\u0026nbsp;noise exposure group and\u0026nbsp;the\u0026nbsp;SS group\u0026nbsp;revealed that the \u0026Delta;GPIAS and \u0026Delta;PPIAS ratios were significantly greater in mice with tinnitus than in those without tinnitus (Fig. 3F and Fig. 4F). Compared with the NE-Tinnitus (-) and control groups, the NE-Tinnitus (+) group had significantly greater \u0026Delta;PPIAS and \u0026Delta;GPIAS ratios (Fig. 3F). However, the average \u0026Delta;GPIAS and \u0026Delta;PPIAS ratios in the SS-Tinnitus (+) group were not different\u0026nbsp;from\u0026nbsp;those in the SS-Tinnitus (-) and control groups\u003cstrong\u003e\u0026nbsp;(Fig. 4F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAlthough both traumatic noise exposure and SS overdose can cause tinnitus, the mice with tinnitus caused by traumatic noise exposure exhibited more interesting behavioral signs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3.\u003c/strong\u003e ∆PPIAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆PPIAS ratio were calculated on BBN, 8kHz, 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. \u003cstrong\u003e(A-E)\u003c/strong\u003e ∆PPIAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). \u003cstrong\u003e(F)\u0026nbsp;\u003c/strong\u003eComparison of average ∆PPIAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean \u0026plusmn; SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001 by two-way ANOVA. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001 by two-way ANOVA. The black hashes represent the statistical difference between the NE-Tinnitus (+) and the NE-Tinnitus (-) group. p \u0026gt; 0.05; #: p \u0026lt; 0.05; ##: p \u0026lt; 0.01; ###: p \u0026lt; 0.001; ####: p \u0026lt; 0.001 by two-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4.\u003c/strong\u003e ∆GAPAS ratio for control (n=10), sodium salicylate injection (n=10), and noise exposure (n=10) group. ∆GAPAS ratio were calculated on 10kHz, 16kHz, 20kHz, 24kHz and 32kHz frequencies. All behavioral tests were taken on day 7 for sodium salicylate group and day 14 for noise exposure group. \u003cstrong\u003e(A-E)\u003c/strong\u003e ∆GAPAS ratio of each mice in NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10). \u003cstrong\u003e(F)\u0026nbsp;\u003c/strong\u003eComparison of average ∆GAPAS ratio among NE-Tinnitus (+) (n=4), NE-Tinnitus (-) (n=6), SS-Tinnitus (+) (n=6), SS-Tinnitus (-) (n=4) and control group (n=10) at different frequencies. Mean \u0026plusmn; SEM is shown in each group. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001 by two-way ANOVA. The green asterisks represent the statistical difference between the NE-Tinnitus (+) and the Control group. p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001 by two-way ANOVA. The black hashes represent the statistical difference between the NE-Tinnitus (+) and the NE-Tinnitus (-) group. p \u0026gt; 0.05; #: p \u0026lt; 0.05; ##: p \u0026lt; 0.01; ###: p \u0026lt; 0.001; ####: p \u0026lt; 0.001 by two-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlterations in ALFF in the SS overdose\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand traumatic noise exposure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egroups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the perspective of energy metabolism, ALFF is defined as the density of neurons with spontaneous synchronized firing in the frequency range of 0.01~0.1 Hz; this value reflects brain region activity (Biswal B 1995; Cordes D 2000; Lowe MJ 1998).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComparing the NE-Tinnitus (+) group with the NE-Tinnitus (-) group, significantly increased ALFF values were observed in the paraflocculus (PFL) and AC (Fig. 5A). However, when the NE-Tinnitus (+) group was compared with the control group, multiple brain regions, including the entorhinal area (ENT), hippocampal\u0026ndash;amygdalar transition area (HATA), supplementary somatosensory area (SSs), striatum-like amygdalar nuclei (sAMY) and medial amygdalar nucleus (MEA), presented decreased ALFF values (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eThe ALFF values in the AC, SSs, and primary somatosensory cortex (SSp) were significantly lower in the SS-Tinnitus (+) group than in the SS-Tinnitus (-) group (Fig. 5C). When the SS-Tinnitus (+) group was compared with the control group, increased ALFF was detected in the anterior olfactory nucleus (AON), whereas decreased ALFF was detected in the medulla (MY) (Fig. 5D).\u003c/p\u003e\n\u003cp\u003eBased on the above results, we believe that SS overdose and traumatic noise exposure may induce tinnitus through different central mechanisms, leading to diverse patterns of ALFF activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.5.\u0026nbsp;\u003c/strong\u003eSignificant enhancement and depression of amplitude of low-frequency fluctuation (ALFF) for noise exposure group and sodium salicylate group respectively. \u003cstrong\u003e(A)\u0026nbsp;\u003c/strong\u003eALFF increases in PFL and AC, NE-Tinnitus (+) group mice versus NE-Tinnitus (-) group mice, at 14th day after traumatic noise exposure.\u003cstrong\u003e\u0026nbsp;(B)\u0026nbsp;\u003c/strong\u003eALFF decreases in ENT, HATA, SSs, sAMY and MEA, NE-Tinnitus (+) group mice versus control mice, at 14th day after traumatic noise exposure.\u003cstrong\u003e\u0026nbsp;(C)\u0026nbsp;\u003c/strong\u003eALFF decreases in AC, SSs, and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. \u003cstrong\u003e(D)\u003c/strong\u003e ALFF changes in MY, pons and AON, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p\u0026lt;0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: PFL, paraflocculus; AC, auditory cortex; ENT, entorhinal area; HATA, hippo-campo amygdalar transition area; SSs, supplementary somatosensory area; sAMY, striatum-like amygdalar nuclei; MEA, medial amygdalar nucleus; CA1, Ammon\u0026rsquo;s horn; AI, agranular insular area; STR, striatum; PAL, pallidum; MOB, main olfactory bulb; MY, medulla; AON, anterior olfactory nucleus; SSp, primary somatosensory cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe alterations of regional homogeneity (ReHo) in overdose sodium salicylate and traumatic noise exposure group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA As a voxel-based measure of brain activity, ReHo is used to evaluate the similarity or synchronization between the time series of a given voxel and its nearest neighbors (Zang et al. 2004), ased on the hypothesis that intrinsic brain activity is manifested by clusters of voxels rather than single voxels. Higher ReHo indicates that neurons in that specific region tend to synchronize and that there is increased consistency of neuronal activity. On the other hand, low ReHo indicates decreased consistency of neuronal activity, which may suggest dysfunction in the brain region (Qu T 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the NE-Tinnitus (+) group, the AC, Ammon\u0026apos;s horn (CA) and striatum (STR) had significantly greater ReHo values than those in the control group \u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6A)\u003c/strong\u003e. However, other brain regions in\u0026nbsp;the\u0026nbsp;NE-Tinnitus (+) group, including the sAMY, HATA, subiculum (SUB), and midbrain (MB),\u0026nbsp;presented decreased ReHo,\u0026nbsp;suggesting a two-way effect of noise exposure. In\u0026nbsp;the\u0026nbsp;NE-Tinnitus (-) group, only decreased ReHo was observed in\u0026nbsp;the\u0026nbsp;HATA, ENT and cortical subplate (CTXsp) regions (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn the SS overdose group, the ReHo exhibited a different pattern of change than that in the traumatic noise exposure group. As shown in \u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6C\u003c/strong\u003e,\u0026nbsp;the\u0026nbsp;SS-Tinnitus (+) group\u0026nbsp;presented\u0026nbsp;lower ReHo values than\u0026nbsp;did the\u0026nbsp;control group in\u0026nbsp;the\u0026nbsp;CA1, AC, SSs, SSp, and retrosplenial area and lateral agranular part (RSPagl).\u0026nbsp;The SS-Tinnitus (-) group\u0026nbsp;presented\u0026nbsp;increased ReHo values in the SSp and CA1 and decreased ReHo values in the hypothalamus (HY) and pons (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6D\u003c/strong\u003e), suggesting that SS administration has an impact on ReHo, particularly\u0026nbsp;in\u0026nbsp;the limbic system, although it fails to induce tinnitus. When\u0026nbsp;the\u0026nbsp;SS-Tinnitus (+) group\u0026nbsp;was compared\u0026nbsp;with the SS-Tinnitus (-) group, multiple regions,\u0026nbsp;including\u0026nbsp;the\u0026nbsp;MY, SSp, SSs, thalamus,\u0026nbsp;sensory‒motor\u0026nbsp;cortex-related (DORsm), hippocampus (HIP), thalamus and polymodal association cortex-related (DORpm)\u0026nbsp;regions,\u0026nbsp;all\u0026nbsp;presented\u0026nbsp;decreased ReHo in tinnitus mice (\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe ALFF and ReHo results in tinnitus mice indicated that different patterns of neural activity developed not only in auditory pathways (such as the AC) but also in limbic systems (such as the hippocampus and thalamus) and somatosensory systems (such as the SSp and SSs). On the other hand, the different brain activity patterns in the SS overdose and traumatic noise exposure groups provide evidence that these two modeling methods may induce tinnitus through different neural circuits or physiological mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.6.\u003c/strong\u003e Significant increase and decrease of regional homogeneity (ReHo) for overdose sodium salicylate and traumatic noise exposure group respectively. (A) ReHo changes in sAMY, HATA, SUB, MB, AC, CA3, and STR, NE-Tinnitus (+) group versus control, at 14th day after traumatic noise exposure. (B) ReHo decreases in ENT, HATA and CTXsp, NE-Tinnitus (-) group mice versus control mice, at 14th day after traumatic noise exposure. (C) ReHo increases in RSPagl, CA1, AC, SSs and SSp, SS-Tinnitus (+) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (D) ReHo changes in HY, pons, SSp and CA1, SS-Tinnitus (-) group mice versus control mice, at 1st day after overdose sodium salicylate injection. (E) ReHo decreases in MY, SSp, DORsm ,SS, HIP, DORpm and SSp, SS-Tinnitus (+) group mice versus SS-Tinnitus (-) group mice, at 1st day after overdose sodium salicylate injection. Statistical analysis threshold is p\u0026lt;0.001 for two sample t-tests. Color heat map scale shows corrected t-values ranging from +10 to 0 and from 0 to -10. Abbreviations: SUB, subiculum; MB, midbrain; CTXsp, cortical subplate; RSPagl, retrosplenial area, lateral agranular; HY, hypothalamus; DORsm, thalamus, sensory-motor cortex related; HIP, hippocampus; DORpm, thalamus, polymodal association cortex related.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlterations in\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;FC patterns in tinnitus mice induced by SS overdose and traumatic noise exposure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFC refers to the similarity between signals arising from two brain regions (Mohanty et al. 2020). Higher FC may indicate that the regions tend to be functionally connected (Greicius et al. 2003; Eickhoff and M\u0026uuml;ller 2015). To investigate whether there were FC changes in tinnitus mice, 7 main brain regions in the auditory pathway were selected as ROIs, and the connectivity between any two regions was examined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn both the traumatic noise exposure group and the SS overdose group, we calculated the FC from 7 brain regions of the central auditory pathway to the AC. In\u0026nbsp;the\u0026nbsp;traumatic noise exposure group, most brain regions, especially\u0026nbsp;the\u0026nbsp;MGB region, presented increased FC in tinnitus mice (\u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e7A and 7C\u003c/strong\u003e). Interestingly, in the SS overdose group,\u0026nbsp;the FC values of the\u0026nbsp;brain regions\u0026nbsp;in the\u0026nbsp;tinnitus group and\u0026nbsp;the\u0026nbsp;control group\u0026nbsp;were not significantly different (\u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e7B and 7C\u003c/strong\u003e). These noticeably different patterns further suggest that noise exposure and sodium salicylate may induce tinnitus through different mechanisms or neural pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 7.\u0026nbsp;\u003c/strong\u003eFC is calculated between the defined AC and related brain regions of interest. (\u003cstrong\u003eA\u003c/strong\u003e) Pattern diagram of related brain regions in correlation with auditory cortex. \u003cstrong\u003e(B)\u003c/strong\u003e FC between NE-Tinnitus (+) group (n=4) and control group (n=10). DCO (NT=0.04\u0026plusmn;0.03，Control=0.11\u0026plusmn;0.03), IC (NT=0.08\u0026plusmn;0.07，Control=0.03\u0026plusmn;0.03),MG (NT=0.09\u0026plusmn;0.06，Control=0.08\u0026plusmn;0.03), NLL (NT=-0.16\u0026plusmn;0.04，Control=0.04\u0026plusmn;0.02), SOC (NT=-0.01\u0026plusmn;0.03，Control=0.05\u0026plusmn;0.03), VOC (NT=0.03\u0026plusmn;0.03，Control=-0.002\u0026plusmn;0.02).\u003cstrong\u003e\u0026nbsp;(C)\u0026nbsp;\u003c/strong\u003eFC between SS-Tinnitus (+) group (n=6) and control group (n=10). DCO (SS=-0.01\u0026plusmn;0.06，Control=0.11\u0026plusmn;0.03), IC (NT=0.09\u0026plusmn;0.11，Control=0.03\u0026plusmn;0.03),MG (NT=0.30\u0026plusmn;0.09，Control=0.08\u0026plusmn;0.03), NLL (NT=-0.16\u0026plusmn;0.04，Control=0.04\u0026plusmn;0.02), SOC (NT=-0.01\u0026plusmn;0.03，Control=0.05\u0026plusmn;0.03), VOC (NT=0.03\u0026plusmn;0.03，Control=-0.002\u0026plusmn;0.02).\u003cstrong\u003e\u0026nbsp;(D)\u0026nbsp;\u003c/strong\u003eComparison of FC among NE-Tinnitus (+) group, SS-Tinnitus (+) group and control group. Mean \u0026plusmn; SEM was shown. :Level of significance is illustrated in the figures with symbols or shaded areas (not significant (n.s.): p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.001). Abbreviations: AC, auditory cortex; DCO, dorsal cochlear nucleus; IC, inferior colliculus; MG, medial geniculate body; NLL, nucleus of the lateral lemniscus; SOC, superior olivary body; VOC, ventral cochlear nucleus.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study systematically\u0026nbsp;demonstrated\u0026nbsp;that mice with tinnitus induced by different methods\u0026nbsp;presented\u0026nbsp;different patterns in hearing threshold shift, behavioral\u0026nbsp;tests\u0026nbsp;and rs-fMRI physiological\u0026nbsp;indices. This study creatively utilized two different methods to induce tinnitus:\u0026nbsp;SS overdose and traumatic noise exposure. In our study, animals exposed to tinnitus-inducing procedures\u0026nbsp;were\u0026nbsp;measured for tinnitus by evaluating how silence modulated their reflexive behavioral responses evoked by unexpected suprathreshold sounds (PPIAS and GPIAS). Many animal studies using PPIAS and GPIAS have used noise\u0026nbsp;exposure levels that induced TTS\u0026nbsp;rather than\u0026nbsp;PTS, similar to our research\u0026nbsp;in which\u0026nbsp;functional hearing was preserved. According to our results, SS overdose could cause\u0026nbsp;a\u0026nbsp;permanent ABR threshold elevation of\u0026nbsp;approximately\u0026nbsp;15 dB SPL\u0026nbsp;at\u0026nbsp;most frequencies,\u0026nbsp;whereas the\u0026nbsp;temporary hearing threshold shift caused by noise exposure\u0026nbsp;returned\u0026nbsp;to normal after 2 weeks. Behavioral tests indicated that both methods successfully induced tinnitus in mice. Mice with tinnitus induced by different methods exhibited different proportions and characteristics, which indicates differences in\u0026nbsp;the\u0026nbsp;underlying mechanisms of tinnitus induction. rs-fMRI data analysis\u0026nbsp;revealed\u0026nbsp;significant\u0026nbsp;increases in the ALFF of the\u0026nbsp;PFL and AC in mice with tinnitus caused by traumatic noise exposure. In\u0026nbsp;the SS\u0026nbsp;overdose group,\u0026nbsp;the\u0026nbsp;ALFF increased in\u0026nbsp;the\u0026nbsp;AON\u0026nbsp;but\u0026nbsp;decreased in\u0026nbsp;the\u0026nbsp;MY and pons. ReHo analysis\u0026nbsp;revealed\u0026nbsp;that neurons in\u0026nbsp;the\u0026nbsp;AC\u0026nbsp;tended\u0026nbsp;to discharge in a more synchronized way in\u0026nbsp;the\u0026nbsp;traumatic noise exposure group, but other brain regions,\u0026nbsp;such as\u0026nbsp;the\u0026nbsp;sAMY, HATA and MGB, presented\u0026nbsp;reduced ReHo values.\u0026nbsp;The tinnitus mice in the SS overdose group also\u0026nbsp;presented\u0026nbsp;increased ReHo values in\u0026nbsp;the\u0026nbsp;AC, as well as in\u0026nbsp;the\u0026nbsp;SSp, SSs, CA1 and RSPagl. This study also calculated the FC between brain regions by exacting time series of fMRI signals in ROIs to identify the\u0026nbsp;sequence\u0026nbsp;and relevance of activated brain regions. Interestingly, in\u0026nbsp;the\u0026nbsp;traumatic noise exposure group, tinnitus mice\u0026nbsp;had\u0026nbsp;significantly lower FC to\u0026nbsp;the\u0026nbsp;NL than\u0026nbsp;did the\u0026nbsp;control group.\u0026nbsp;Tinnitus\u0026nbsp;mice in\u0026nbsp;the\u0026nbsp;SS group\u0026nbsp;presented\u0026nbsp;significantly\u0026nbsp;greater\u0026nbsp;FC to\u0026nbsp;the\u0026nbsp;MGB than\u0026nbsp;did those in the\u0026nbsp;control group. Based on ALFF, ReHo, and FC analyses,\u0026nbsp;the\u0026nbsp;two methods may induce tinnitus in different ways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTinnitus induced by traumatic noise exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNoise-related factors are responsible for a significant percentage of tinnitus\u0026nbsp;cases. In nearly 58% of cases, individuals subjected to acute acoustic trauma develop symptoms of tinnitus as a result of the immediate damage to the inner ear\u0026nbsp;(Bhatt et al. 2016). It is widely accepted that noise exposure affects neural plasticity in the central nervous system. Increasing evidence suggests that chronic tinnitus can result from a failure of limbic structures to block hyperactive auditory signals induced by noise exposure\u0026nbsp;(Rauschecker et al. 2010; Zhang et al. 2021). Noise-induced tinnitus is prevalent in humans and animals with normal audiograms because of increased neuronal activity caused by noise exposure and plasticity\u0026nbsp;(Nagaraj et al. 2020; Qu et al. 2019). In individuals claiming compensation for work-related hearing loss, the prevalence of tinnitus was approximately constant over a wide hearing range\u0026nbsp;(Lewkowski et al. 2023). Some authors have suggested that noise may cause tinnitus, and even tinnitus patients with normal audiograms may have limited cochlear damage. There is also evidence that not all patients with hearing loss develop tinnitus(Alshabory et al. 2022; Pavlidis et al. 2023). Our study used a level of traumatic noise exposure that induced TTS rather than PTS, similar to previous studies in mice and guinea pigs in which functional hearing was preserved\u0026nbsp;(Longenecker and Galazyuk 2016; Park et al. 2020). Many studies suggest that the differences in peripheral damage and central plasticity among individual animals are due to the heterogeneity of tinnitus pathology\u0026nbsp;(Hickox and Liberman 2014; Knipper et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTinnitus induced by SS overdose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSS inhibits cyclooxygenase activity, which could disrupt the conversion of arachidonic acid into prostaglandin H2.\u0026nbsp;Excessive\u0026nbsp;SS could increase the\u0026nbsp;probability of\u0026nbsp;channel opening in\u0026nbsp;the\u0026nbsp;NMDA receptor\u0026nbsp;(Yamakura and Shimoji 1999). SS\u0026nbsp;can\u0026nbsp;cause tinnitus by overactivating NMDA receptors, resulting in neuronal\u0026nbsp;hyperexcitation\u0026nbsp;and tonotopic shifts in auditory pathways\u0026nbsp;(Ruel et al. 2008; Jiang et al. 2017). Extracellular recordings in vitro have indicated that salicylate can drastically alter the spontaneous firing rate (SFR) of neurons in the MGB, although the direction of change is complex. Approximately 52.4% of neurons increased their firing rate after SS treatment, whereas\u0026nbsp;firing rates decreased in approximately 47.6% of neurons\u0026nbsp;(Basta et al. 2008; Ma et al. 2006). Since the MGB provides excitatory inputs to the primary AC (A1), changes in the MGB are likely to significantly impact activity in A1.\u0026nbsp;The preceding results demonstrated that\u0026nbsp;salicylate\u0026nbsp;not only\u0026nbsp;suppresses\u0026nbsp;the neural output of the peripheral auditory system but also alters activity in the hCNS\u0026nbsp;(Stolzberg et al. 2011). I In some cases, SFRs in A1 and the anterior auditory field (AAF) decreased slightly after salicylate treatment,\u0026nbsp;whereas\u0026nbsp;those in\u0026nbsp;the secondary AC (A2)\u0026nbsp;increased.\u0026nbsp;A1 neurons receive afferent inputs\u0026nbsp;mainly\u0026nbsp;from the lemniscal pathway,\u0026nbsp;and A2 neurons receive afferent information from the extralemniscal pathway\u0026nbsp;(Eggermont and Kenmochi 1998). The reduction in\u0026nbsp;the\u0026nbsp;A1 SFR after SS treatment may therefore be due\u0026nbsp;primarily\u0026nbsp;to the suppression of neural output from the cochlea and classical auditory pathway,\u0026nbsp;whereas the enhanced spontaneous firing\u0026nbsp;observed\u0026nbsp;in A2 may reflect the changes occurring at both auditory and non-auditory loci in the CNS\u0026nbsp;(Huang and Winer 2000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePPIAS and GPIAS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnlike humans, in whom tinnitus can be self-reported, animal models face exceptional challenges in reliably inducing and detecting tinnitus. There are also complex variations in the perception of tinnitus depending on background noise, stress, fatigue, and other factors\u0026nbsp;(Clifford et al. 2019). PPIAS and GPIAS are reliable methods for the detection of tinnitus. In the salicylate tinnitus model, GPIAS typically decreases in the low-frequency range compared with the range between 8 and 16 kHz\u0026nbsp;(Berger et al. 2013). The GPIAS deficiency in the salicylate model usually returns to normal within 72 hours after the last administration, so we conducted the appropriate tests within 24 hours after the last administration of SS in our study\u0026nbsp;(Stolzberg et al. 2012). However, the prerequisite for normal hearing limits the usage opportunities for GPIAS. Hearing loss caused by noise exposure or ototoxic drugs can make background sound and the embedded gaps less audible(Fournier and H\u0026eacute;bert 2013). Therefore, when hearing loss is induced in one ear to trigger tinnitus, the other ear should be protected. To ensure the accuracy \u0026nbsp; of PPIAS and GPIAS, we protected the right ear of the mice in the traumatic noise exposure group using an earplug.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAC and tinnitus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSalicylate-induced tinnitus leads to reduced SFRs in the AC\u0026nbsp;(Nore\u0026ntilde;a et al. 2010). Our results also revealed decreased synchronization and neuronal density with spontaneous synchronized firing in the AC induced by SS overdose. However, a significant increase in metabolic activity in the AC and inferior colliculus was detected via micropositron emission tomography in an animal model, potentially reflecting increased inhibitory synaptic activity\u0026nbsp;(Paul et al. 2009). The reduction\u0026nbsp;in\u0026nbsp;SFR in\u0026nbsp;the\u0026nbsp;AC, as well as the increase in the\u0026nbsp;SFRs\u0026nbsp;of the second auditory cortical field and external nucleus of the IC,\u0026nbsp;suggest\u0026nbsp;that while the AC is involved in hyperacusis-like effects, the extralemniscal pathway is the primary cause of salicylate-induced tinnitus(Manabe et al. 1997). Our findings also strongly suggested that traumatic noise exposure could cause increased cortical reorganization, SFRs and interneuronal synchrony in\u0026nbsp;the\u0026nbsp;AC,\u0026nbsp;which\u0026nbsp;is\u0026nbsp;similar to\u0026nbsp;the findings of\u0026nbsp;previous\u0026nbsp;studies\u0026nbsp;(Eggermont 2015). As determined behaviorally by conditioned response testing, the AC could exhibit increased SFRs as a result of downregulation of inhibition in the tinnitus region according to Yang et al.\u0026rsquo;s research in a traumatic noise animal model\u0026nbsp;(Yang et al. 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMGB and tinnitus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MGB is a mandatory relay station along the auditory pathway that mediates the thalamocortical network involved in tinnitus (Chen et al. 2015). Classical and non-classical ascending auditory pathways contribute differently to auditory stimuli processing in the MGB and are most likely to contribute to tinnitus pathophysiology (Pickles 2015). The evidence across several species indicates that the MGB dynamically shapes simple tones and complex vocalizations before auditory sensations reach the cerebral cortex (Mihai et al. 2019; Kraus et al. 1994; Cai et al. 2016). Previous studies have shown that animals with tinnitus have an increased number of spikes per burst and tonic GABAA currents, which leads to increased output from the MGB to higher auditory cortices (Sametsky et al. 2015). In a tinnitus model induced by SS, when assessing the coherence between the MGB and PAC, SS enhanced coherence in the gamma band, which was suggested to be a direct neural correlate of tinnitus, influencing thalamocortical networks (Vianney-Rodrigues et al. 2019). However, spontaneous firing rates in the MGB have been found to be unaffected in rats with tinnitus induced by acoustic noise trauma (Barry et al. 2019). Like in previous studies, MGB excitability was observed only in mice with SS overdose-induced tinnitus and not in mice with traumatic noise exposure-induced tinnitus in our study (Brinkmann et al. 2021). According to neuroimaging studies on tinnitus patients, there is decreased FC between the thalamus and superior frontal gyrus and increased spontaneous neural activity between the two areas (Zhang et al. 2015; Chen et al. 2014). The contribution of the MGB to tinnitus can possibly be explained by the fact that unpleasant auditory inputs are normally \u0026quot;cancelled out\u0026quot; at the level of the MGB. However, the noise cancellation mechanism in patients with tinnitus is dysfunctional, resulting in disinhibition of the MGB, which may contribute to the perception of tinnitus sounds (Rauschecker et al. 2010; Elgoyhen et al. 2015).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, this study aimed to investigate the neural basis of tinnitus by comparing two commonly used animal models: traumatic noise exposure and SS overdose. Both methods were found to successfully induce tinnitus in mice, as evidenced by behavioral tests. However, rs-fMRI analysis revealed distinct patterns of neural activity in tinnitus mice from each group, suggesting differences in the underlying mechanisms of tinnitus induction. These findings suggest that traumatic noise exposure and SS overdose induce tinnitus through distinct neural mechanisms involving different neural circuits and pathways. Understanding these differences is crucial for developing targeted therapies for tinnitus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Natural Science Foundation of Shanghai No 19ZR1408700, Shanghai 2020 \u0026quot;Science and Technology Innovation Action Plan\u0026quot;- \u0026quot;One Belt One Road\u0026quot; International Cooperation Project (20410740600), Clinical Research Plan of SHDC (SHDC2020CR1049B), General Project of Shanghai Hongkou District Health Commission（2102-21）and the Key Clinical Specialty Construction Project of Shanghai Hongkou District (HKLCZD2024A03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions: conceptualisation:\u0026nbsp;\u003c/strong\u003eW.Z., J.N. \u003cstrong\u003edata curation:\u0026nbsp;\u003c/strong\u003eW.Z., J.N, Y.Z, R.M. \u003cstrong\u003esoftware:\u003c/strong\u003e W.Z., J.N.\u003cstrong\u003e\u0026nbsp;formal analysis:\u0026nbsp;\u003c/strong\u003eW.Z., J.N, Y.Z, R.M. \u003cstrong\u003evisualisation:\u0026nbsp;\u003c/strong\u003eW.Z., J.N., J.Y. \u003cstrong\u003emethodology:\u0026nbsp;\u003c/strong\u003eM.C. \u003cstrong\u003ewriting (original draft):\u0026nbsp;\u003c/strong\u003eW.Z., J.S. \u003cstrong\u003esupervision:\u003c/strong\u003e M.C., J.Y. \u003cstrong\u003efunding acquisition:\u0026nbsp;\u003c/strong\u003eW.Z., J.S., M.C., J.Y. \u003cstrong\u003ewriting (review and editing):\u0026nbsp;\u003c/strong\u003eM.C., J.Y.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e This study was approved by the Ethics Committee of the Eye Ear Nose \u0026amp; Throat Hospital of Fudan University (No.2021-2021220). All the experimental animals were treated in accordance with the guidelines of the Ethical Board of EENT Hospital, Fudan University.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdjamian P, Hall DA, Palmer AR, Allan TW, Langers DRM (2014) Neuroanatomical abnormalities in chronic tinnitus in the human brain. Neuroscience and Biobehavioral Reviews 45:119-133. doi:10.1016/j.neubiorev.2014.05.013\u003c/li\u003e\n \u003cli\u003eAlshabory HF, Gabr TA, Kotait MA (2022) Distortion Product Otoacoustic Emissions (DPOAEs) In Tinnitus Patients. Int Arch Otorhinolaryngol 26 (1):e046-e057. doi:10.1055/s-0040-1722248\u003c/li\u003e\n \u003cli\u003eAuerbach BD, Rodrigues PV, Salvi RJ (2014) Central gain control in tinnitus and hyperacusis. Frontiers In Neurology 5:206. doi:10.3389/fneur.2014.00206\u003c/li\u003e\n \u003cli\u003eBarry KM, Robertson D, Mulders WHAM (2019) Changes in auditory thalamus neural firing patterns after acoustic trauma in rats. 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Neural Plast 2021:8833087. doi:10.1155/2021/8833087\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Functional MRI, Noise, Sodium Salicylate, Tinnitus, auditory cortex, medial geniculate body","lastPublishedDoi":"10.21203/rs.3.rs-5014033/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5014033/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tinnitus, a phantom auditory sensation, significantly impacts quality of life and social interactions. While central gain changes, including hyperactivity and increased neural synchrony, have been implicated in tinnitus, the underlying neural mechanisms remain unclear. This study utilized resting-state functional magnetic resonance imaging (rs-fMRI) to investigate the neural basis of tinnitus induced in mice by two distinct methods, traumatic noise exposure and sodium salicylate overdose. Behavioral tests confirmed the successful induction of tinnitus in both groups. rs-fMRI analysis revealed distinct patterns of neural activity in mice with tinnitus compared with control mice. Traumatic noise exposure induced an increase in amplitude of low-frequency fluctuations (ALFF) in the paraflocculus and auditory cortex, as well as a decrease in regional homogeneity (ReHo) in limbic system regions. On the other hand, sodium salicylate overdose led to decreased ALFF and ReHo in the auditory cortex, somatosensory system and limbic system regions. Functional connectivity analysis further highlighted differences between the two models, with traumatic noise exposure affecting connectivity with the inferior colliculus and sodium salicylate overdose affecting connectivity with the medial geniculate body. These findings suggest that traumatic noise exposure and sodium salicylate overdose induce tinnitus through distinct neural mechanisms, potentially involving different neural circuits and pathways. Further research is needed to explore these mechanisms and develop targeted therapies for tinnitus.","manuscriptTitle":"Resting-state Functional MRI Study of Mice with Tinnitus Induced by Traumatic Noise Exposure and Sodium Salicylate Overdose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-22 09:12:46","doi":"10.21203/rs.3.rs-5014033/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8bf062e-7a8b-4e40-8d5d-70286f7f2e68","owner":[],"postedDate":"October 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-27T15:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-22 09:12:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5014033","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5014033","identity":"rs-5014033","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}