Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet

Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet

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Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet Yoo Yeon Kim a, 1, Janet Ren Chao b, Chulho Kim c, Boyoung Kim a, Phuong Thi-Thanh Nguyen a, Harry Jung d, Jiwon Chang e, Jun Ho Lee d, e, **, 1, Jun Gyo Suh a, * a

Department of Medical Genetics, College of Medicine, Hallym University, Chuncheon, 24252, Republic of Korea Division of Otolaryngology, Department of Surgery, Yale School of Medicine, New Haven, CT, 06510, USA Department of Neurology, Hallym University College of Medicine, Chuncheon, 24252, Republic of Korea d Institute of New Frontier Research Team, Hallym University College of Medicine, Chuncheon, 24252, Republic of Korea e Department of Otorhinolaryngology-Head and Neck Surgery, Hallym University College of Medicine, Chuncheon, 24252, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2019 Accepted 21 December 2019 Available online xxx

Age-related hearing loss (ARHL) is a neurodegenerative disease associated with an aged population. ARHL is influenced by biological factors such as aging, sex difference, and atherosclerosis. The mechanisms of ARHL caused by atherosclerosis have not been previously determined in apolipoprotein E knockout (ApoE KO) male mice. To investigate the onset and cause of the hearing loss, ApoE KO male mice were treated with a western diet (ApoE KO-WD) for 16 weeks. The lipid profile, atherosclerotic plaques throughout the aorta, and auditory brainstem response (ABR) thresholds were measured in the ApoE KO-WD male mice. The expression of S100 calcium-binding protein B (S100B), a neuronal damage biomarker, was also observed. Reactive oxygen species (ROS) and apoptosis rates were detected in the cochlea of the ApoE KO male mice. Atherosclerotic plaques on the aorta and ABR thresholds were significantly increased in the ApoE KO-WD male mice at 24 weeks of age. ABR thresholds had a statistically significant positive correlation with the area of atherosclerotic plaques (r ¼ 0.783, p ¼ 0.013) in male mice at 24 weeks of age. S100B protein expression and the dihydroethidium (DHE) reaction to ROS in the cochlear spiral ganglion neurons (SGNs) were significantly increased in the ApoE KO and ApoE KOWD male mice. Cells positive for active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) in the SGNs were significantly increased in ApoE KO-WD male mice indicating an increased rate of cellular apoptosis. In conclusion, ROS in the SGNs were activated by increased S100B expression in ApoE KO-WD male mice, and this resulted in an increased apoptosis rate. Thus, hearing loss began at 16 weeks in ApoE KO-WD male mice. Our results suggest that the ApoE KO-WD male mice are a suitable animal model for studying ARHL associated with exacerbated atherosclerosis. © 2020 Elsevier Inc. All rights reserved.

Keywords: Reactive oxygen specie Apoptosis Spiral ganglion neurons Hearing loss Atherosclerosis

1. Introduction Age-related hearing loss (ARHL), or presbycusis, is the progressive hearing loss associated with age. It is a neurodegenerative * Corresponding author. Department of Medical Genetics, College of Medicine, Hallym University, 1, Hallymdaehak-gil, Chuncheon, Gangwon-do, 24252, Republic of Korea. ** Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, Hallym University College of Medicine, Hallym University, 1, Hallymdaehak-gil, Chuncheon, Gangwon-do, 24252, Republic of Korea. E-mail addresses: [email protected] (J.H. Lee), [email protected] (J.G. Suh). 1 Laboratory of Brain and Cognitive Sciences for Convergence Medicine, Hallym University College of Medicine, Anyang, 14054, Republic of Korea.

disease and affects hundreds of millions of people worldwide [1]. The incidence of ARHL increases dramatically in patients over 50 years of age in the USA as shown in the study of Bainbridge and Wallhagen [2] where 15%, 31%, and 63% of patients were affected at 50e59, 60e69, and over 70 years of age respectively. As life expectancy increases, it is estimated that the number of people with ARHL will double by 2031 [2]. ARHL is classified into four types of pathological lesions: loss of sensory hair cells (sensory hearing loss), spinal ganglion neurons (SGNs) loss (neuronal hearing loss), atrophy of the stria vascularis (metabolic hearing loss), and changes in the basilar membrane stiffness (cochlear conductive hearing loss). Most cases of ARHL develop as a result of mixed pathological alterations [3e6].

https://doi.org/10.1016/j.bbrc.2019.12.100 0006-291X/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100

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ARHL is caused by genetic, biological, and environmental factors, including noise exposure, ototoxicity, infections, hyperlipidemia, atherosclerosis, and diabetes [7e9]. Abnormal total cholesterol level is a major risk factor for atherosclerosis and therefore impacts neurodegenerative diseases through its effect on regulation of neuron survival and function [10]. The pathophysiological mechanisms of sensorineural hearing loss are associated with oxidative stress, excitotoxicity, inflammation, and apoptosis of SGNs [11,12]. S100 calcium-binding protein B (S100B) is produced primarily in astrocytes and Schwann cells and is commonly used as an indicator of neuronal damage [13,14]. A high concentration of S100B in neurons produces reactive oxygen species (ROS) utilizing a receptor for advanced glycation endproducts (RAGE). The increase of ROS will activate caspase-3 and accelerate apoptosis in neurons including the SGNs [15,16]. Inbred strain (C57BL/6J) is a suitable animal model for ARHL research. Auditory brainstem response (ABR) thresholds in these mice are higher in females than in males after 6 months of age [17,18]. This sex-based variation is also reported in humans. The prevalence of ARHL is higher in men at 20e39 years of age compared to women in that age group [19]. Shuster et al. suggest that estrogen is closely related to ABR thresholds because hearing loss occurs rapidly in post-menopausal women [20]. The apolipoprotein E knockout (ApoE KO) mouse serves as an animal model for the association of hyperlipidemia and atherosclerosis with hearing loss [21,22]. Atherosclerosis is associated with alterations of the cochlear morphology and resultant hearing loss in ApoE KO female mice fed an atherogenic diet, as a result of the stenosis of the spiral modiolar artery (SMA) and low endothelial nitric oxide synthase (eNOS) activity [22]. Since hearing loss in mice shows variation based on sex [17,18], we investigated hearing loss in ApoE KO male mice fed a western diet (ApoE KO-WD). ABR thresholds and lipid profiles were measured in these mice, and atherosclerotic plaques on the aorta and apoptosis in the SGNs were observed. Our study demonstrated that the collective area of atherosclerotic plaques was associated with increased hearing loss in ApoE KO male mice. Additionally, the determined reason for hearing loss in ApoE KO-WD male mice was apoptosis of SGNs and cells within the stria vascularis. The onset and causes of hearing loss in ApoE KO male mice were therefore determined to be different from those of ApoE KO female mice, indicating that auditory functioning in ApoE KO-WD mice show sex-based variation.

2.2. Measurement of lipid profiles Retro-orbital plexus plasma was obtained from the mice after 12 h of fasting at 8, 16, and 24 weeks. Lipid profiles (total-cholesterol, HDL-cholesterol, LDL-cholesterol, and triglyceride) were measured using standard blood chemistry equipment (Kornelab20XT, Thermo Fisher Scientific, USA). 2.3. Atherosclerotic plaques staining with Sudan Ⅳ The aorta was isolated from the aortic arch to the aortic iliac bifurcation in mice at 24 weeks of age. Each aorta was opened longitudinally and pinned flat in a black wax Petri dish, and then incubated overnight in a 4% paraformaldehyde-sucrose solution at room temperature. The aorta was then stained for 10 min with 5% Sudan IV (Sigma, USA) in 100% acetone and 70% ethanol (1:1). After that, the aorta was decolorized twice for 3 min each time with 70% ethanol and then washed with 1X PBS for 5 min to remove the ethanol. For each sample, the area of the stained atherosclerotic plaques was measured relative to the area of the whole aorta by image J software (National Institute of Health, USA). Three samples per group were used for the intensity analysis. 2.4. Auditory brainstem response (ABR) test To evaluate the auditory function, ABR thresholds were measured in all mice at 8, 16, and 24 weeks of age. Anesthesia was performed with 3% isoflurane mixed with oxygen by an RC2þ anesthesia system (Lab. Inc., USA). The ABR was recorded via electrodes placed under the skin of the anesthetized mice. The electrodes were located at the vertex in the midline of the scalp (active), below the pinna of the left and right ears (reference), and in the tail (ground). The scalp electrodes were adjusted to keep the impedance below 25 kU using an EPA PreAmplifier (Interacoustics, Denmark). Earphones were inserted and auditory stimulation was provided as click sounds at 2e4 kHz generated using ECLIPS (Interacoustics, Denmark). The output was recorded on an OtoAccess software (ver. 1.4, Interacoustics, Denmark). Sound responses were recorded at decibel peak equivalent sound pressure levels (dBpeSPL) in the left and right ears. The stimulus duration was 8 ms at a rate of 19.3 stimuli per second. Auditory thresholds were determined by increasing the sound intensity of the click stimulus from 30 to 130 dBpeSPL. 2.5. Detection of ROS and apoptosis in the cochlea

2. Materials and methods 2.1. Animals and diets All 6-week-old male mice, C57BL/6J (B6, DBL Co., Korea) and B6.129P2-Apoe tm1Unc/J (ApoE KO, Jackson Laboratory, USA), were fed a chow diet (Research diets Inc., USA) containing 10 kcal % fat and 0.0014% cholesterol for 2 weeks. At the age of 8 weeks, the ApoE KO mice were randomly assigned to one of two groups: Group 1 (n ¼ 12) was continuously fed a chow diet (ApoE KO-CD), and group 2 (n ¼ 12) was fed a western-type diet (ApoE KO-WD) containing 41 kcal % fat and 0.125% cholesterol (Research diets Inc., USA) over the following 16 weeks. The control group (n ¼ 18) consisted of B6 mice that were fed a chow diet (B6-CD) for the following 16 weeks. The mice were kept in a specific-pathogen-free animal care facility that maintained an environment at 22 ± 2  C with a 55 ± 10% relative humidity and a 12 h light and 12 h darkness routine cycle. The animal study was conducted in accordance with the regulations of the Institutional Animal Care and Use Committees of Hallym University (Hallym 2016e31).

The cochlea was removed from the 24-week-old mice and fixed in 4% paraformaldehyde for 1 day. The cochlea was incubated in decalcifying solution-lite (Sigma, USA) for 5 h at RT. The tissue was then dehydrated in graded alcohols and embedded in paraffin wax. The paraffin-embedded cochlear tissues were made into 8-mm thick slides. After the deparaffinization and dehydration processes were complete, antigen retrieval was performed on the tissue. The sections were treated with 1X Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 10% horse serum for 1 h and incubated with the primary antibody anti-S100-beta (1:200, Abcam, UK), anti-caspase-3 (1:100, Abcam, UK), and anti-beta III tubulin (1:1,000, Abcam, UK) overnight at 4  C. Alexa 488conjugated goat-anti-rabbit (1:500, Abcam, UK) and Alexa 647conjugated donkey-anti-mouse (1:500, Life Technologies, USA) were used as the secondary antibody. To observe the reactive oxygen species (ROS), the tissues were incubated with 10 mM of dihydroethidium (DHE, Invitrogen, USA) in a light-protected area at 37  C for 30 min. After that, 40 ,6-diamidino-2-phenylindole (DAPI, 1:1000, Invitrogen, USA) stained the tissues at RT for 3 min. The

Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100

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terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed utilizing the DeadEnd™ Fluorometric TUNEL System (Promega, USA). Photographs were taken of the left and right ear at the middle turn of the cochlea using confocal microscopy (Carl Zeiss, LSM 710, Germany). The expression intensity was measured using ZEN (ZEN 2 PRO, Carl Zeiss, Germany) and image J software (National Institute of Health, USA). Three to five samples per group were used for the intensity analysis. 2.6. Statistical analysis All data were expressed as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) was used for multiple comparisons. The correlation between ABR thresholds, the percentage of atherosclerotic plaques, and the lipid profiles was performed by a simple linear regression analysis. The correlation between the independent variable and the dependent variable was signified by a correlation coefficient (r), and the proportion of the variance predicted dependent variable was expressed as a coefficient of determination (r2). Statistical significance was accepted at

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P < 0.05. All data were analyzed by the SPSS statistics 22.0 (IBM Corp., USA). 3. Results 3.1. Exacerbation of atherosclerosis and hyperlipidemia in the ApoE KO-WD male mice Atherosclerotic plaques were measured on the inner surface throughout the aorta using Sudan Ⅳ staining. Atherosclerotic plaques throughout the aorta were significantly increased in the ApoE KO-WD mice compared with the ApoE KO-CD mice and the B6-CD mice at 24 weeks of age (Fig. 1A). Atherosclerotic plaques were located mainly on the ascending aorta, right and left common carotid arteries, and common iliac artery. Total-cholesterol (TC), LDL-cholesterol (LDL-Chol), and HDLcholesterol (HDL-Chol) were significantly increased in the ApoE KO-WD mice compared to the ApoE KO-CD mice and the B6-CD mice at 16 and 24 weeks of age (Fig. 1B). Triglyceride (TG) levels in the ApoE KO-WD mice and ApoE KO-CD mice were increased

Fig. 1. Atherosclerotic plaques on the aorta and lipid profile. (A) Representative en face images show Sudan Ⅳ-stained atherosclerotic plaques on the aortas. Atherosclerotic plaques are represented as the percentage of plaques relative to the total aorta area (n ¼ 3). (B) Changes of lipid profile with age. C57BL/6J mice were fed a chow diet (B6-CD), and ApoE KO mice were fed a chow diet (ApoE KO-CD) or a western diet (ApoE KO-WD) for 16 weeks.

Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100

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compared to the B6-CD mice at 8, 16, and 24 weeks of age. These results demonstrate that hyperlipidemia was more severe in the male ApoE KO-WD mice than in the ApoE KO-CD mice after 16 weeks of age. In summary, atherosclerotic plaques were dramatically increased in male ApoE KO mice fed a western diet for 16 weeks.

3.2. Progressive hearing loss in the ApoE KO-WD male mice ABR thresholds were compared in each group (B6-CD, ApoE KOCD, and ApoE KO-WD) at 8, 16, and 24 weeks of age. ABR thresholds in the left ear of the B6-CD mice were significantly increased from 24 weeks of age. However, there was no difference in the right ear of B6-CD mice throughout the experimental period (Fig. 2A). ABR thresholds in ApoE KO-CD mice were significantly increased from 16 weeks of age compared to 8 weeks in both ears (Fig. 2B). In ApoE KO-WD mice, ABR thresholds in both ears were significantly increased from 16 weeks of age. Furthermore, ABR thresholds in the left ear of the ApoE KO-WD mice were significantly elevated at 24 weeks of age compared to those at 16 weeks of age (Fig. 2C). The ABR thresholds of the left ear at 8, 16, and 24 weeks of age were 41,76, and 96 dBpeSPL respectively in the ApoE KO-WD mice. These results indicate that the ApoE KO-WD mice had progressive hearing

loss. When ABR thresholds were compared among 3 groups (B6-CD, ApoE KO-CD, and ApoE KO-WD), there were no significant differences at 8 weeks and 16 weeks of age. At 24 weeks of age, the ABR thresholds in the left ear were significantly elevated in the ApoE KO-WD mice compared with those of the ApoE KO-CD mice and the B6-CD mice (Fig. 2D). There was no significant difference in the ABR thresholds in the right ear among the 3 groups at any period. These findings demonstrate that the hearing loss of the left ear was more severe in the ApoE KO-WD mice in than those of other groups at 24 weeks of age.

3.3. Association of ABR thresholds with atherosclerotic plaques and lipid profiles in the ApoE KO-WD male mice There was a statistically significant positive correlation between ABR thresholds and atherosclerotic plaques (r ¼ 0.783, p ¼ 0.013) in male mice at 24 weeks of age (Table 1). In addition, the correlation analysis showed that ABR thresholds were significantly correlated with TC (r ¼ 0.568, p ¼ 0.000), LDL-Chol (r ¼ 0.562, p ¼ 0.000), HDL-Chol (r ¼ 0.564, p ¼ 0.000), and TG (r ¼ 0.325, p ¼ 0.038) in male mice at 24 weeks of age. As a result, the increase of ABR thresholds was associated with atherosclerotic plaques and

Fig. 2. Measurement of auditory brainstem response (ABR) thresholds. ABR thresholds of the left ear were compared among three groups at 24 weeks of age (D).

Table 1 Simple linear regression analysis of auditory brainstem response (ABR) thresholds with atherosclerotic plaques, total cholesterol (TC), low-density lipoprotein cholesterol (LDLChol), high-density cholesterol (HDL-Chol), and triglycerides (TG). Independent variables

Atherosclerotic plaques (%) TC (mg/dl) LDL-Chol (mg/dl) HDL-Chol (mg/dl) TG (mg/dl)

ABR Thresholds (dBpeSPL) Correlation coefficient, r

Coefficient of determination, r2

P value

0.783 0.568 0.562 0.564 0.325

0.613 0.323 0.318 0.316 0.106

0.013* <0.001*** <0.001*** <0.001*** 0.038*

*P < 0.05, ***P < 0.001.

Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100

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hyperlipidemia in male mice at 24 weeks of age. 3.4. Hearing loss influenced by apoptosis in the SGNs of the ApoE KO-WD male mice Expression of S100B was significantly increased in the SGNs of the ApoE KO-WD mice and ApoE KO-CD mice compared to the B6CD mice at 24 weeks of age (Fig. 3A). ROS were detected by the DHE assay and were significantly increased in the ApoE KO-WD mice and ApoE KO-CD mice than that of B6-CD mice at 24 weeks of age (Fig. 3B). To investigate the impact of increased ROS on apoptosis prevalence in the SGNs, apoptosis was detected by the TUNEL assay and active caspase-3 in each group (B6-CD, ApoE KO-CD, and ApoE KO-WD) at 24 weeks of age. Apoptotic cells in the SGNs detected by the TUNEL assay were significantly increased in the ApoE KO-WD mice compared to those in the ApoE KO-CD mice and B6-CD mice (Fig. 4A). The intensity of active caspase-3, an apoptosis marker localized in the cytoplasm of the SGNs, was significantly increased in the ApoE KO-WD mice compared to the ApoE KO-CD mice and the B6-CD mice (Fig. 4B). In conclusion, hearing loss in ApoE KOWD male may be associated with apoptosis in the SGNs. 4. Discussion This study investigated the onset and causes of hearing loss in the ApoE KO-WD male mice. ABR thresholds in the ApoE KO-WD male mice were significantly increased after 16 weeks of age (Fig. 2). ABR thresholds in the ApoE KO-WD male mice were significantly increased compared to the ApoE KO-CD male mice and

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the B6-CD male mice at 24 weeks of age (Fig. 2). Hyperlipidemia in the ApoE KO-WD male mice was aggravated from 16 weeks of age (Fig. 1B). The increased atherosclerotic plaques in the ApoE KO-WD male mice was significant at 24 weeks of age (Fig. 1A). The elevation of ABR thresholds in these mice was positively correlated with atherosclerotic plaques and hyperlipidemia (Table 1). The hearing loss in the ApoE KO-WD male mice was associated with apoptosis (Fig. 4) induced by increased ROS in the SGNs (Fig. 3B). In these studies, ABR thresholds in the ApoE KO-WD male mice were significantly higher than those of the ApoE KO-CD male mice and the B6-CD male mice at 24 weeks of age. However, there was no difference of ABR thresholds between the ApoE KO-CD male mice and the B6-CD male mice at the same age (Fig. 2). Guo et al. [22] reported that ABR thresholds in the ApoE KO-WD female mice were significantly higher than the ApoE KO-CD female mice at 24 weeks of age. In addition, ABR thresholds in the ApoE KO-CD female mice also were significantly elevated compared to the B6-CD female mice at the same age [22]. Caligiuri et al. [23] observed that atherosclerotic lesions on the aorta of the ApoE KO female mice at 16 weeks of age were increased compared to the males at the same age. However, there was no difference in the size of the atherosclerotic plaques in the female mice compared to the male mice 48 weeks of age possibly due to the decreased estrogen levels in the female mice at that age [23,24]. For these reasons, atherosclerosis in the ApoE KO male mice may have been less developed than that of the ApoE KO female mice at 24 weeks of age. ABR thresholds in the ApoE KO-WD female were higher than the ApoE KO-WD male mice because atherosclerosis in ApoE KO-WD female was more severe than in the ApoE KO-WD male mice at 24 weeks of age.

Fig. 3. Expression of S100B and reactive oxygen species (ROS) in the spiral ganglion neurons (SGNs). (A) S100B (green), beta-3 tubulin (TUBB3; red), and DAPI (blue) were stained in the SGNs at 24 weeks of age (upper images). The relative intensity of S100B was measured in the SGNs area (lower graph). (B) Dihydroethidium (DHE; red) and DAPI (blue) were stained in the SGNs at 24 weeks of age (upper images). The relative intensity of DHE was measured in the SGNs area (lower graph). Scale bar is 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100

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Fig. 4. Detection of apoptosis and active caspase-3 in the spiral ganglion neurons (SGNs). (A) TUNEL (green) and propidium iodide (PI; red) were stained in the SGNs at 24 weeks of age (upper images). The relative intensity of TUNEL/PI in the SGNs was calculated using Image J software (lower graph). (B) Active caspase-3 (green) and beta3-tubulin (TUBB3; red) were stained in the SGNs at 24 weeks of age (upper images). Relative intensity of active caspase-3/TUBB3 was measured in the SGNs area (lower graph). Scale bar, 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Endothelial dysfunction, one of the indicators of atherosclerosis, is induced by increased ROS and decreased endothelial nitric oxide synthase in the ApoE KO mice [21]. However, little was previously known about the relationship between increased ROS and the cochlear dysfunctions in ApoE KO mice. In this study, expression of S100B was significantly increased in SGNs of the ApoE KO-WD male mice (Fig. 3A), ROS were also significantly increased as a result of the high concentration of S100B (Fig. 3B). The increased ROS increased induction of apoptosis dramatically in SGNs (Fig. 4), and associated early-onset hearing loss in the ApoE KO-WD male mice resulted (Fig. 2C). Since hearing loss in the ApoE KO-WD female mice was found to be caused by apoptosis in the stria vascularis [25], we measured ROS and observed associated apoptosis in this area. ROS were significantly increased in the ApoE KO-WD male mice compared to the ApoE KO-CD male mice and the B6-CD male mice at 24 weeks of age (Fig. S1A). Apoptosis was significantly increased in the ApoE KO- CD and the ApoE KO-WD (Fig. S1B). Some researchers insisted that decreased nitric oxide bioavailability in the stria vascularis and SGNs was the main cause of hearing loss in the ApoE KO-WD female mice [22,25]. Hearing loss of the ApoE KOWD male mice was therefore determined to be caused by apoptosis in SGNs and the stria vascularis. This was due to increased ROS pathways and associated apoptosis in the SGNs as a result of increased S100B. In conclusion, hearing loss in the ApoE KO-WD male mice started at 16 weeks of age and degenerated up until 24 weeks of age. This hearing loss was caused by apoptosis following increased ROS in SGNs and stria vascularis as a result of increasing of S100B. Our results indicate that the ApoE KO-WD male mice are a useful

model to investigate relationship between age-related hearing loss and atherosclerosis. Funding This work was supported by grants (NRF2016R1D1A1B01014128, NRF-2016R1D1A2B02011858) through the National Research Foundation of Korea (NRF). Declaration of competing interest All authors have seen and approved manuscript. The authors declare that there is no financial conflict of interests to publish these results. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.100. References [1] R.D. Frisina, B. Ding, X. Zhu, J.P. Walton, Age-related hearing loss: prevention of threshold declines, cell loss and apoptosis in spiral ganglion neurons, Aging 8 (2016) 2081e2099, https://doi.org/10.18632/aging.101045. [2] K.E. Bainbridge, M.I. Wallhagen, Hearing loss in an aging American population: extent, impact, and management, Annu. Rev. Public Health 35 (2014) 139e152, https://doi.org/10.1146/annurev-publhealth-032013-182510. [3] G.A. Gates, J.H. Mills, Presbycusis, Lancet 366 (2005) 1111e1120, https:// doi.org/10.1016/S0140-6736(05)67423-5. [4] J.B. Nadol Jr., Schuknecht: "presbycusis, The Laryngoscope 65 (1995) 402e419,

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Please cite this article as: Y.Y. Kim et al., Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.100