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Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice
Neurochemistry International 133 (2020) 104649
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Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice
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Zheng-De Dua, Shu-Guang Hana, Teng-Fei Qua, Bin Guoa, Shu-Kui Yua, Wei Weib, Shuai Fengc, Ke Liua,∗∗, Shu-Sheng Gonga,∗ a
Department of Otorhinolaryngology, Beijing Friendship Hospital, Capital Medical University, 95 Yongan Road, Xicheng District, Beijing, 100050, China Department of Otology, Shengjing Hospital, China Medical University, 36 Sanhao Street, Heping District, Shenyang, 110004, China c Department of Otorhinolaryngology, The First Hospital of China Medical University, 155 Nanjingbei Street, Heping District, Shenyang, 110000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Presbycusis Cochlear ribbon synapsis Mitochondria Oxidative damage D-galactose
Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.
1. Introduction Presbycusis, also known as age-related hearing loss, results from age-related degeneration of the cochlea and the central auditory system (Howarth and Shone, 2006). Since physiological aging processes in animals and humans develop very slowly and the genetic and environmental backgrounds of humans are heterogeneous, an investigation of presbycusis in healthy organisms is very limited. The chronic administration of D-galactose (D-gal) can rapidly induce aging in rodents. Rodents treated with D-gal exhibit increased oxidative damages, declined mitochondrial functions, weakened immune responses, and attenuated learning and memory (Kumar et al., 2009; Lu et al., 2010;
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Rehman et al., 2017). These symptoms reflect those in physiological aging well. Therefore, D-gal-induced aging in rodents is commonly used in research as a model of aging and age-related diseases. In exposed to D-gal-induced aging, a previous study reported that only a small number of marginal cells in the stria vascularis of the cochlea (Du et al., 2012), as well as very few neurons in the central auditory system (Du et al., 2015; Du et al., 2019a; Sun et al., 2015; Zeng et al., 2014), are lost because of the activation of mitochondria-dependent apoptosis. However, research rarely systematically reports the changes of hair cells, spiral ganglion cells (SGCs), and ribbon synapses between the inner hair cells (IHCs) and SGCs in the cochlea of rodents with D-galinduced aging.
Corresponding author. Corresponding author. E-mail addresses: [email protected] (K. Liu), [email protected] (S.-S. Gong).
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https://doi.org/10.1016/j.neuint.2019.104649 Received 20 October 2019; Received in revised form 15 December 2019; Accepted 19 December 2019 Available online 20 December 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved.
Neurochemistry International 133 (2020) 104649
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10% solution of sodium EDTA for 3 d at 4 °C. The cochleae of one side were further treated for cochlear frozen sections; those of the contralateral side were used for cochlear surface preparations. After decalcification, the cochleae used for frozen sections were incubated overnight at 4 °C in 100 mM sodium phosphate buffer containing 30% sucrose and placed afterwards in optimal cutting temperature-embedding compound (Leica Microsystems, Germany). Sections (10 μm) from cryofixed tissues were collected on 3-aminopropyl-trimethoxysilanecoated slides (Sigma-Aldrich) and dried for 2 h in preparation for the H &E staining or immunohistochemical analysis. The cochlear sections stained with H&E were examined under a light microscope (Nikon, Tokyo, Japan). The Rosenthal's canal was divided into three regions: apex, middle, and base. In each mouse, we evaluated every third modiolar section obtained from 1 cochlea for a total of 10 sections. Tissues from the same animals were used for SGC counting.
In this study, we used the D-gal-induced aging model in mice to determine changes in the numbers of outer hair cells (OHCs), IHCs, SGCs, and ribbon synapses and to observe its influence on the morphology of hair cell stereocilia. We also measured the levels of oxidative stress, oxidative damage to mitochondrial DNA, mitochondrial DNA mutation, and ATP production in the cochlea. The present study may further uncover D-gal-induced morphological changes in the cochlea and potential causative mechanisms of presbycusis. 2. Material and methods 2.1. Animals and D-gal administration Sixty 5-week-old male C57BL/6J mice were obtained from the Experimental Animal Centre of Capital Medical University. The mice were housed in a temperature-controlled (20–22 °C) room and had free access to food and drinking water. After acclimation for a week, the mice were randomly divided into three groups (n = 20 per group) depending on the administered D-gal (Sigma-Aldrich, USA) dose as follows: Control group, the mice were injected subcutaneously with 0.9% saline (the vehicle of D-gal) once a day for 6 weeks; D-gal-L (lowdose D-gal) group, the mice were injected subcutaneously with 500 mg/ kg D-gal once a day for 6 weeks; and D-gal-H (high-dose D-gal) group, the mice were injected subcutaneously with 1000 mg/kg D-gal once a day for 6 weeks. After the measurement of auditory functions, the mice were killed, and both sides of the cochleae were dissected for the determination of the H2O2 content, total superoxide dismutase (T-SOD) activity, and ATP levels, as well as for the extraction of genomic DNA, total RNA, and protein. Alternatively, the cochleae of one side were perfused with 4% paraformaldehyde for cochlear sections and surface preparations, whereas the cochleae of the contralateral side were perfused with 2.5% glutaraldehyde for scanning electron microscopy. All protocols conformed to the Guideline for the Care and Use of Laboratory Animals of the National Institutes of Health. The study protocols were approved by the Committee on the Ethics of Animal Experiments of Capital Medical University.
2.4. Immunohistochemistry of cochlear surface preparations The numbers of OHCs, IHCs, and ribbon synapses were counted using the cochlear surface preparation (n = 4 per group) technique (Han et al., 2016; Kujawa and Liberman, 2009). After decalcification, the softened otic capsule, stria vascularis, tectorial membrane, and Reissner's membrane were removed under an anatomy microscope. The remaining cochlear sensory epithelium was permeabilized in 0.3% Triton X-100 solution for 1 h at room temperature. The specimens were then washed three times with phosphate-buffered saline and blocked with 10% goat serum for 1 h at room temperature. The specimens were incubated with monoclonal mouse anti-carboxyl-terminal binding protein 2 (CtBP2) IgG1 (diluted 1:400; BD Biosciences, USA) and polyclonal rabbit anti-myosin VIIa IgG (diluted 1:400; Abcam, USA) primary antibodies overnight at 4 °C. After washing three times, the specimens were incubated with Alexa Fluor 568- and Alexa Fluor 594conjugated secondary antibodies at a concentration of 1:300 at room temperature for 2 h in darkness. After a final wash, the cochlear sensory epithelia were divided into three segments (apical, middle, and basal turn) and mounted on slides with mounting medium containing 4′,6diamidino-2-phenylindole (DAPI; ZSGB-BIO, China). DAPI-labeled OHCs, myosin VIIa-labeled IHCs, and CtBP2-labeled synaptic ribbons were then examined with a laser scanning confocal microscope (TCS SP8; Leica, Germany). Survival rates of OHCs and IHCs were quantitively evaluated by counting residual hair cells in the cochlea. Synaptic ribbons were normalized to the number of IHCs in the cochlea.
2.2. Auditory function evaluation The auditory function of mice was evaluated by auditory brainstem response (ABR) (Someya et al., 2010). A total of 18 mice (n = 6 per group) were anesthetized by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture. The sound delivery tube of an inserted earphone was tightly fitted into the external auditory canal; subdermal needle electrodes were inserted at the vertex (active), right mastoid (reference), and left mastoid (ground). ABR responses were measured with a tone burst stimulus (3 ms duration, 1 ms rise/fall times, at a rate of 21.1/s) at 8 kHz (low frequency), 16 kHz (middle frequency), and 32 kHz (high frequency) using the TDT System 3 (Tucker-Davis Technologies, Alachua, FL, USA) starting from a 90-dB sound pressure level (SPL) and decreasing in 5-dB increments to the acoustic threshold. At each frequency, determine the ABR threshold, which refers to the minimal SPL resulting in a reliable ABR recording with one or more distinguishable waves that can be clearly identified by visual inspection. It is necessary to repeat the process for low SPLs around the threshold to ensure the consistency of the waveforms. The threshold was independently determined by two researchers who were blinded to the group assignment.
2.5. Immunohistochemistry of cochlear frozen sections The oxidative damage to mitochondrial DNA was evaluated by 8hydroxy-2-deoxyguanosine (8-OHdG) immunohistochemical analysis (Du et al., 2019a). The cochlear sections were washed and incubated with 0.3% Triton X-100 solution for 30 min at room temperature. They were washed again and blocked with 10% goat serum for 1 h at room temperature. Next, slides were incubated with monoclonal mouse anti8-OHdG IgG2b (diluted 1:200; Abcam), monoclonal mouse anti-β-tubulin III IgG2a (diluted 1:100; Abcam), and polyclonal rabbit antimyosin VIIa IgG (diluted 1:400; Abcam) primary antibodies overnight at 4 °C. Slides were then washed and incubated with Alexa Fluor 568-, Alexa Fluor 488-, and Alexa Fluor 594-conjugated secondary antibodies at a concentration of 1:300 at room temperature for 2 h in darkness. After a final wash, sections were mounted with mounting medium containing DAPI (ZSGB-BIO). DAPI-labeled nuclei, 8-OHdG-labeled DNA damage, myosin VIIa-labeled hair cells, and β-tubulin III-labeled SGCs and neurofilaments were captured using a laser scanning confocal microscope (Leica TCS SP8). The expression of 8-OHdG was analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., USA). In the negative control, sections were treated in the same manner, except that incubation with primary antibody was omitted.
2.3. Hematoxylin and eosin (H&E) staining The general morphology of the cochlea and the number of SGCs were analyzed by H&E staining (Someya et al., 2009). Twelve mice (n = 4 per group) were decapitated, and the cochleae of both sides were removed from the temporal bones and fixed in 4% paraformaldehyde overnight at 4 °C. The cochleae were then decalcified in a 2
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used to amplify the mtDNA 3860-bp deletion and 12S rRNA were previously described (Zhang et al., 2009, 2013) and as follows: 3860-bp deletion: forward, 5′-TCATTCTAGCCTCGTACCAACA-3′, reverse, 5′-GAGGTCTGGGTCATTTTCGTTA-3’; 12S rRNA: forward, 5′-ACCGCG GTCATACGATTAAC-3′, reverse, 5′-CCCAGTTTGGGTCTTAGCTG-3’. PCR amplification was performed on a StepOne Real-Time PCR System (Applied Biosystems, USA) in a 25-μl reaction volume consisting of 12.5 μl of 2 × SYBR PCR mix (Tiangen Biotech Co.), 1.0 μl each of forward and reverse primers (7.5 μM), 2.5 μl of sample DNA (20 ng/μl), and 8.0 μl of distilled water. The cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The cycle number at which a significant increase in normalized fluorescence was first detected was designated as the threshold cycle number (Ct). The ratio of mtDNA 3860-bp deletion to total mtDNA was calculated as ΔCt = (Ct3860-bp deletion − Ct12S rRNA), and the relative expression was calculated as 2−ΔΔCt. The PCR products of the mtDNA 3860-bp deletion were cloned and verified using an ABI Prism 377XL sequencer (Applied Biosystems, USA).
2.6. Scanning electron microscopy The ultrastructure of hair cell stereocilia was observed using scanning electron microscopy (Marie et al., 2018). Twelve mice (n = 4 per group) were decapitated, and their cochleae were prepared as described for the H&E staining procedure. After exposing the cochlear sensory epithelium, it was dehydrated in a graded series of ethanol, criticalpoint dried in CO2, coated with gold palladium, and observed using a scanning electron microscope (S-4800; Hitachi Science System, Ltd., Japan). 2.7. Protein extraction and western blot analysis The levels of NADPH oxidase 2 (NOX2) and uncoupling protein 2 (UCP2) protein expression were determined by western blot analysis in 12 mice (n = 4 per group). After decapitation, the cochleae were rapidly removed bilaterally from the temporal bones. The soft tissues were then harvested from the cochleae under an anatomy microscope and homogenized in a RIPA Lysis Buffer (Beyotime, China) according to the manufacturer's instructions. Total protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime). Of each protein lysate, 25 μg were separated by 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After incubating the membranes in a blocking solution (5% nonfat dry milk in Tris-buffered saline containing 1% Tween-20 [TBST]) for 1 h at room temperature, they were washed briefly in TBST and incubated with anti-NOX2 (diluted 1:500; Abcam), anti-UCP2 (diluted 1:500; Abcam), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; diluted 1:1000; Cell Signaling Technology, USA) primary antibodies overnight at 4 °C. After three additional washing steps in TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted 1:5000; ZSGB-BIO) for 1 h at room temperature. Finally, the membranes were visualized in a darkened room using BeyoECL Plus (Beyotime). The detected bands were quantified using the Image-Pro Plus 6.0 software. GAPDH was used as an internal control.
2.10. Statistical analysis The data are presented as the mean ± standard deviation (SD). Statistical tests were performed with SPSS 13.0 software (SPSS Inc., USA). Statistical significance was analyzed via independent-samples ttests or one-way ANOVA. The independent-samples t-test was used to evaluate differences between the Control, and D-gal-H groups. The least significant difference (LSD) post hoc-test in ANOVA was used to evaluate differences among the Control, D-gal-L, and D-gal-H groups. Differences with a P-value < 0.05 were considered to be statistically significant. 3. Results 3.1. Auditory impairment induced by D-gal The auditory functions of mice in the Control group and the D-gal groups were evaluated using the ABR test. In mice, ABR waves consist of five vertex-positive peaks, and wave I reflects the function of cochlear ribbon synapses. We found that the ABR waves I-IV of mice exposed to D-gal-induced aging displayed a poorer peak morphology than those of control mice (Fig. 1A). In the Control, D-gal-L, and D-galH groups, the mean ABR thresholds of mice were 27 ± 4.08 dB SPL, 30 ± 10.00 dB SPL, and 31 ± 9.17 dB SPL at 8 kHz (low frequency), respectively; 33 ± 5.16 dB SPL, 36 ± 3.76 dB SPL, and 38 ± 5.24 dB SPL at 16 kHz (middle frequency), respectively; and 52 ± 6.06 dB SPL, 54 ± 6.65 dB SPL, and 58 ± 7.58 dB SPL at 32 kHz (high frequency), respectively. There were no significant differences among the three groups in all frequencies examined (P > 0.05, Fig. 1B). In the Control, D-gal-L, and D-gal-H groups, the mean amplitudes of the ABR wave I at 90 dB SPL were 1.15 ± 0.42 μV, 0.70 ± 0.30 μV, and 0.53 ± 0.18 μV at 8 kHz, respectively; 0.91 ± 0.27 μV, 0.67 ± 0.20 μV, and 0.53 ± 0.13 μV at 16 kHz, respectively; and 0.55 ± 0.09 μV, 0.40 ± 0.11 μV, and 0.31 ± 0.08 μV at 32 kHz, respectively. Compared with control mice, the mean amplitude of the ABR wave I was in D-gal-treated mice significantly decreased in all frequencies (P < 0.05 or P < 0.01, Fig. 1C). At 90 dB SPL, the latency values of the ABR wave I in mice of the Control, D-gal-L, and D-gal-H groups were 1.97 ± 0.09 ms, 2.02 ± 0.15 ms, and 2.23 ± 0.29 ms at 8 kHz, respectively; 1.96 ± 0.10 ms, 2.04 ± 0.17 ms, and 2.06 ± 0.22 ms at 16 kHz, respectively; and 2.16 ± 0.22 ms, 2.47 ± 0.49 ms, and 2.71 ± 0.46 ms at 32 kHz, respectively. Only in mice of the D-gal-H group, the ABR wave I latency was significantly increased at 8 kHz and 32 kHz (both P < 0.05) in comparison to control mice, whereas no significant differences were detected between the Control group and the D-gal-L group (Fig. 1D).
2.8. H2O2, T-SOD activity, and ATP measurements Twelve mice (n = 4 per group) were sacrificed, and the cochleae of both sides were rapidly removed from the temporal bones. The soft tissues were then harvested from the cochleae under an anatomy microscope and homogenized in cold saline. The homogenate was centrifuged at 4000×g for 15 min at 4 °C, and the supernatant was used for H2O2, T-SOD activity, and ATP measurements. Protein concentrations were evaluated using the Enhanced BCA Protein Assay Kit (Beyotime). H2O2, T-SOD activity, and ATP levels in the cochleae of different groups were quantified using colorimetric kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. These results are representative of at least three separate experiments. 2.9. DNA isolation and mitochondrial DNA (mtDNA) 3860-bp deletion assay The accumulation of mtDNA with 3860-bp deletion was evaluated in 12 mice (n = 4 per group) using a real-time PCR assay (Zhang et al., 2009, 2013). After rapid removal of the bilateral cochleae from the temporal bones, the soft tissues of the cochleae were harvested under an anatomy microscope for genomic DNA isolation. The DNA was extracted using a Genomic DNA Isolation kit (Tiangen Biotech Co., China) according to the manufacturer's instructions. The DNA concentration of each specimen was measured with the GeneQuant pro DNA/RNA Calculator (Amersham Pharmacia Biotech, Sweden). Due to the rarity of the deletion, the 12S rRNA gene was used as an internal control for the murine mitochondrial genome (GenBank: NC_006914). The primers 3
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Figure 1. Auditory function parameters in the Control, D-gal-L, and D-gal-H groups. (A) Representative ABR traces in mice of the Control and the D-gal-H groups at 8 kHz & 90 dB SPL. (B) Mean values of the ABR threshold in the Control, D-gal-L, and D-gal-H groups. (C) Mean amplitudes of the ABR wave I in the Control, D-gal-L, and D-gal-H groups at 90 dB SPL. (D) Mean latency values of the ABR wave I in the Control, D-gal-L, and D-gal-H groups at 90 dB SPL. Data are expressed as the mean ± SD of 6 mice per group. *P < 0.05 or **P < 0.01 versus the Control group.
IHCs were rarely lost in both the Control and the D-gal-H group.
3.2. Tissue morphology in the cochlea of mice presenting D-gal-induced aging
3.3. D-gal-induced insult to cochlear ribbon synapses
The general cochlea morphology was observed in H&E-stained cochlear sections. The cochlea contains an apical, middle, and basal turn (Fig. 2A); the organ of Corti contains in every turn one row of IHCs and three rows of OHCs (Fig. 2C). No loss of IHCs, OHCs, or SGCs was discovered in the cochlear sections of the D-gal-H group (Fig. 2B–D). The number of DAPI-labeled OHCs in cochlear surface preparations (Fig. 2E) was not significantly different between the Control and the Dgal-H group (Fig. 2F). Additionally, the stereocilia of IHCs and OHCs were observed using scanning electron microscopy. Although the stereocilia of OHCs in the D-gal-H group showed mild flattening compared with those in the Control group (Fig. 2G), the stereocilia of OHCs and
The numbers of IHCs and ribbon synapses were also counted in cochlear surface preparations. The IHC ribbon, labeled by CtBP2, is the presynaptic structure of ribbon synapses, and it also allows a suitable estimate for the number of auditory nerve fibers (Fig. 3A). The numbers of myosin VIIa-labeled IHCs were not significantly different among the Control, D-gal-L, and D-gal-H groups (P > 0.05; Fig. 3B, D). CtBP2labeled IHC ribbons were visible on the basal side of the IHCs (Fig. 3B). In the Control, D-gal-L, and D-gal-H groups, the mean numbers of synaptic ribbons per IHC were 14.75 ± 0.96, 12.50 ± 0.58, and 10.75 ± 1.50 in the apical turn of the cochlea, respectively; Fig. 2. Cochlear tissue morphology in mice of the Control group and the D-gal-H group. (A) Representative image of a cochlear section from the D-gal-H group. (B) SGCs in the middle turn of a cochlea from the D-gal-H group. (C) The organ of Corti (OC) in the middle turn of a cochlea from the D-gal-H group. (D) Mean numbers of SGCs in the Control group and the D-gal-H group. (E) Representative images of DAPI-labeled OHCs (blue) in the basal turn of a cochlea. (F) OHC survival rates in the Control and D-gal-H groups. (G) Representative images of hair cell stereocilia in the basal turn of a cochlea under a scanning electron microscope. Data are expressed as the mean ± SD of 4 mice per group. Scale bars: A, 500 μm; B, C, E, 50 μm; G, 5 μm.
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Fig. 3. Numbers of IHCs and synaptic ribbons in the Control, D-gal-L, and D-gal-H groups. (A) Schematic of the cochlear ribbon synapses showing an IHC (blue) and its afferent innervation as it appears in the cochlea immunostained for synaptic ribbon protein (CtBP2: red) and auditory nerve fibers (NF: green). (B) Representative cochlear surface preparation images of CtBP2-labeled synaptic ribbons (red) and myosin VIIa-labeled IHCs (blue) in the basal turn of cochleae from the Control, D-gal-L, and D-gal-H groups. (C) Representative cochlear section images of β-tubulin III-labeled NF (green), myosin VIIa-labeled IHCs (blue), and DAPIlabeled nuclei (purple) in the basal turn of cochleae from the Control, D-gal-L, and D-gal-H groups. (D) IHC survival rate in the apical, middle, and basal turn of cochleae from mice of the Control, D-gal-L, and D-gal-H groups. (E) Synaptic ribbons per IHC in the apical, middle, and basal turn of cochleae from the Control, D-galL, and D-gal-H groups. Data are expressed as the mean ± SD of 4 mice per group. *P < 0.05 or **P < 0.01 versus the Control group, ^P < 0.05 versus the D-gal-L group. Scale bars: B, 50 μm; C, 100 μm. Fig. 4. NOX2-and UCP2-mediated oxidative stress in cochleae of the Control, D-gal-L, and D-gal-H groups. (A) Representative images of NOX2 and UCP2 expression in cochleae of the Control, D-gal-L, and D-gal-H groups using western blotting. (B) Relative protein expression of NOX2 and UCP2 in cochleae of the Control, D-gal-L, and D-gal-H groups. (C) H2O2 levels in cochleae of the Control, D-gal-L, and D-gal-H groups. (D) T-SOD activity levels in cochleae of the Control, D-gal-L, and D-gal-H groups. *P < 0.05 or **P < 0.01 versus the Control group, ^^P < 0.01 versus the D-gal-L group.
middle, and basal turn of the cochlea in comparison to those in the Dgal-L group (P < 0.05, Fig. 3E). To further evaluate the D-gal-induced insult to ribbon synapses, the auditory nerve fibers were examined in immunohistochemical staining using cochlear sections. As shown in Fig. 3C, the positive expression of β-tubulin III was in the SGCs, auditory nerve fibers between SGCs and IHC, and non-neuronal cells lining the inner sulcus. Although the morphological characteristics of β-
16.25 ± 0.96, 12.50 ± 1.29, and 10.50 ± 1.29 in the middle turn, respectively; and 13.50 ± 1.29, 11.25 ± 0.96, and 8.25 ± 1.71 in the basal turn, respectively. Compared with the Control group, the synaptic ribbons per IHC of the D-gal-L group exhibited a statistically significant decrease in the apical, middle, and basal turn of the cochlea (P < 0.05 or P < 0.01, Fig. 3E). Similarly, the numbers of synaptic ribbons in the D-gal-H group were significantly reduced in the apical, 5
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tubulin III-labeled nerve fibers did not allow an accurate quantification of fibers per IHC, the decreased density of β-tubulin III-positive elements in both the D-gal-L and the D-gal-H group was obvious in qualitative assessments (Fig. 3C).
IHCs and SGCs induced by D-gal. Compared with the Control group, the expression of 8-OHdG in IHCs of the D-gal-L and the D-gal-H group was significantly increased by the factor of 3.33 ± 0.31 and 9.65 ± 1.54, respectively (both P < 0.01, Fig. 5A and B), whereas the 8-OHdG expression in SGCs of the D-gal-L group and the D-gal-H group was increased 2.10 ± 0.38-fold and 2.77 ± 0.42-fold, respectively (both P < 0.01; Fig. 5A, C).
3.4. Oxidative stress in the cochlea of mice exposed to D-gal-induced aging The NADPH oxidase system and mitochondria can generate reactive oxygen species (ROS) (Bedard and Krause, 2007; Hiona and Leeuwenburgh, 2008). Therefore, the protein levels of NOX2 and UCP2 were evaluated using western blot analyses. Compared with the Control group, NOX2 protein levels in the D-gal-L and D-gal-H groups were 1.83 ± 0.16-fold and 4.70 ± 0.64-fold (P < 0.05 and P < 0.01, respectively) increased, whereas UCP2 protein levels in those groups were significantly increased by a factor of 1.64 ± 0.21 and 2.68 ± 0.35, respectively (both P < 0.01; Fig. 4A and B). To further investigate the oxidative stress in the cochlea of D-gal-treated mice, we measured the levels of H2O2 and T-SOD activity. H2O2 levels in the Control, D-gal-L, and D-gal-H groups were 14.91 ± 1.07 mmol/g protein, 24.52 ± 3.60 mmol/g protein, and 34.50 ± 1.66 mmol/g protein, respectively. The statistical analysis revealed that the H2O2 levels in the D-gal-L group and in the D-gal-H group were significantly higher than that in the Control group (both P < 0.01, Fig. 4C). Moreover, the T-SOD activity levels in the Control, D-gal-L, and D-gal-H groups were 128.00 ± 6.10 U/mg protein, 94.13 ± 4.59 U/mg protein, and 76.78 ± 7.91 U/mg protein, respectively. Similar to the results in the H2O2 measurements, the T-SOD activity levels in both the D-gal-L group and the D-gal-H group were significantly decreased in comparison to the Control group (both P < 0.01, Fig. 4D).
3.6. Accumulation of the mtDNA 3860-bp deletion and decline in ATP levels in the cochlea of D-gal-treated mice The levels of mtDNA mutation in the cochleae of mice exposed to Dgal-induced aging were evaluated by detecting the mtDNA 3860-bp deletion, also known as the age-related common deletion (CD) (Du et al., 2019b). Using real-time PCR, the relative CD levels in the cochleae of the D-gal-L group and the D-gal-H group were significantly increased by the factor of 4.69 ± 1.13 and 8.86 ± 2.69, respectively, in comparison to that of the Control group (P < 0.05 and P < 0.01, respectively; Fig. 6A). The PCR product sequences identified two putative breakage/fusion sites between the two 15-bp repeat (AGCCCTA CTAATTAC) (at 9089–9103 or 12963–12978) and confirmed that the deleted region was 3860 bp long (Fig. 6C). To further evaluate mitochondrial functions, we measured the levels of ATP production using a colorimetric kit. The levels of ATP production in the Control, D-gal-L, and D-gal-H groups were 12.65 ± 0.50 nmol/mg protein, 9.47 ± 0.61 nmol/mg protein, and 7.38 ± 0.77 nmol/mg protein, respectively. The ATP levels in the D-gal-L and D-gal-H groups were significantly decreased compared to those in the Control group (Fig. 6B). 4. Discussion
3.5. Oxidative damage to mtDNA in the murine cochlea after D-gal-induced aging
The current study is the first demonstrating that the cochlear IHC ribbon synapses are the main insult site at early stages of D-gal-induced aging in mice, although marginal cell apoptosis in the stria vascularis of the cochlea has been previously described by Du et al. (Du et al., 2012). Ribbon synapses between IHCs and SGCs are formed as the first afferent
As shown in Fig. 5A, the expression of 8-OHdG, a marker of oxidative DNA damage (Prabhulkar and Li, 2010), was mainly localized in the cytoplasm of IHCs and SGCs of the D-gal-L and D-gal-H groups, indicating an increased oxidative DNA damage in the mitochondria of
Figure 5. 8-OHdG expression in IHCs and SGCs of cochleae from mice of the Control, D-gal-L, and Dgal-H groups. (A) Representative images of 8-OHdG (red) in myosin VIIa-labeled IHCs (green) and β-tubulin III-labeled SGCs (green) of the Control, D-gal-L, and D-gal-H groups. (B) Relative 8-OHdG expression in IHCs of the Control group, the D-gal-L group, and the D-gal-H group. (C) Relative 8-OHdG expression in SGCs of the Control, D-gal-L, and D-gal-H groups. Data are expressed as the mean ± SD of 4 mice per group. **P < 0.01 versus the Control group, ^P < 0.05 or ^^P < 0.01 versus the D-gal-L group. Scale bar: 50 μm.
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Fig. 6. CD accumulation and decline in ATP levels in cochleae of the Control, D-gal-L, and D-gal-H groups. (A) The relative amount of CD in cochleae of the Control, D-gal-L, and D-gal-H groups. (B) Levels of ATP production in cochleae of the Control group, the D-gal-L group, and the D-gal-H group. (C) Validation of CD by sequencing. The arrows point to the putative breakpoint sites between the two underlined 15-bp direct repeat sequence 5′-AGCCCTA CTAATTAC-3' (at 9089–9103 or 12963–12978). Data are expressed as the mean ± SD of 4 mice per group. *P < 0.05 or **P < 0.01 versus the Control group, ^^P < 0.01 versus the D-gal-L group.
in D-gal-treated mice, the density of auditory nerve fibers from SGCs was largely decreased, and this was accompanied by increased mitochondrial oxidative damage in SGCs, suggesting that mitochondria in SGCs have an impact on the auditory nerve fibers from these cells. Taken together, the oxidative damage to mitochondrial DNA and the subsequent dysfunctions in IHCs and SGCs might cause age-related insults to pre- and postsynaptic structures of the cochlear ribbon synapse, respectively. According to the mitochondrial aging theory (Hiona and Leeuwenburgh, 2008), mitochondrial DNA is highly susceptible to oxidative damage during aging because of excess ROS production, the decline in antioxidants, and the lack of protective histones. Du et al. (Du et al., 2012). demonstrated that the NADPH oxidase system is the main source of ROS production in the cochlea of rats exposed to D-gal-induced aging. NOX2, also known as gp91phox, is widely distributed in different cells and tissues (Bokoch and Knaus, 2003), including the cochlea (Kim et al., 2010). NOX2 constitutes with its corresponding subunits a functional enzyme complex that transfers electrons from NADPH to oxygen to generate ROS(Bedard and Krause, 2007). In the current study, we also found NOX2 protein overexpressed in the cochlea of D-gal-treated mice. This indicates that NOX2-generated ROS might be, at least partly, responsible for D-gal-induced mitochondrial DNA damage in IHCs and SGCs. Apart from the NADPH oxidase system, mitochondria are another important source of ROS production in vivo (Hiona and Leeuwenburgh, 2008). Mitochondrial ROS generation can activate UCP2, which is located in the inner mitochondrial membrane (Divakaruni and Brand, 2011; Donadelli et al., 2014; Toda and Diano, 2014). Therefore, the UCP2 expression can indirectly reflect cellular ROS levels. Previous studies demonstrated UCP2 overexpression in the cochlea and the central auditory system of rats exposed to D-gal-induced aging (Du et al., 2014, 2012). In the present study, we also found that the levels of UCP2 protein were significantly increased in the cochlea of D-gal-treated mice, suggesting that mitochondrial ROS may also be involved in the D-gal-induced damage to mitochondrial DNA in IHCs and SGCs.
neuronal connection in the auditory nervous system (Glowatzki and Fuchs, 2002; Meyer et al., 2009). A previous study also found significant synaptic losses long before a decline in ABR threshold values or hair cell counts were detected in early aging stages of CBA/CaJ mice (Sergeyenko et al., 2013). ABR thresholds have been shown to be insensitive to widespread loss of cochlear IHCs ribbon synapses that can occur after noise exposure or at the early stage of aging processes, while the amplitudes of the ABR wave I, which reflect the function of ribbon synapses, were largely decreased (Kujawa and Liberman, 2009; Sergeyenko et al., 2013). Our findings demonstrate that at early stages of D-gal-induced aging, the ABR threshold in mice is not significantly different, but the amplitude and latency of the ABR wave I are significantly decreased and increased, respectively. This indicates that the impairment of the ABR wave I is correlated with synaptic losses. These results further demonstrate that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis. The cochlear ribbon synapse is characterized by a high rate of neurotransmitter release (Griesinger et al., 2005). The effective neurotransmitter transport system in IHCs for the maintenance of synaptic vesicles recycling and tonic exocytosis is largely dependent on ATP produced by mitochondria (Kang et al., 2008; Li et al., 2004; Stowers et al., 2002; Verstreken et al., 2005). In this study, we observed increased oxidative damage to mitochondrial DNA in the IHCs, as well as CD in the cochlea of D-gal-treated mice. Furthermore, we also demonstrated a decrease in mitochondrial ATP production in the cochlea of mice exposed to D-gal-induced aging. These outcomes indicate that mitochondrial oxidative damage and subsequent dysfunctions might be responsible for the impairment of presynaptic structures at the cochlear ribbon synapse. Previously published studies suggest that an age-related loss of auditory nerve fibers, especially low-spontaneous rate fibers, might be an important contributor to age-related hearing loss (Liberman, 1982; Schmiedt et al., 1996). Based on an electron-microscopic study using serial sections of the cat cochlea, the low-spontaneous rate fibers are susceptible to injury because of impaired mitochondria (Liberman, 1980). In the present study, we also found that 7
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In conclusion, this study is the first demonstrating that the cochlear ribbon synapse is the primary insult site in the D-gal-induced aging model and that mitochondrial oxidative damage mediated by the NADPH oxidase system and mitochondria might be responsible for this insult. Physiological aging in the cochlea can be experimentally modeled by D-gal administration, and D-gal-induced aging in mice is an ideal animal model to investigate mechanisms and interventional strategies of age-related insults to cochlear ribbon synapses in early stages of presbycusis.
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Schmiedt, R.A., Mills, J.H., Boettcher, F.A., 1996. Age-related loss of activity of auditorynerve fibers. J. Neurophysiol. 76, 2799–2803. Sergeyenko, Y., Lall, K., Liberman, M.C., Kujawa, S.G., 2013. Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J. Neurosci. 33, 13686–13694. Someya, S., Xu, J., Kondo, K., Ding, D., Salvi, R.J., Yamasoba, T., Rabinovitch, P.S., Weindruch, R., Leeuwenburgh, C., Tanokura, M., Prolla, T.A., 2009. Age-related hearing loss in C57BL/6J mice is mediated by Bak-dependent mitochondrial apoptosis. Proc. Natl. Acad. Sci. U.S.A. 106, 19432–19437. Someya, S., Yu, W., Hallows, W.C., Xu, J., Vann, J.M., Leeuwenburgh, C., Tanokura, M., Denu, J.M., Prolla, T.A., 2010. Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction. Cell 143, 802–812. Stowers, R.S., Megeath, L.J., Górska-Andrzejak, J., Meinertzhagen, I.A., Schwarz, T.L., 2002. Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein. Neuron 36, 1063–1077. Sun, H.Y., Hu, Y.J., Zhao, X.Y., Zhong, Y., Zeng, L.L., Chen, X.B., Yuan, J., Wu, J., Sun, Y., Kong, W., Kong, W.J., 2015. Age-related changes in mitochondrial antioxidant enzyme Trx2 and TXNIP-Trx2-ASK1 signal pathways in the auditory cortex of a mimetic aging rat model: changes to Trx2 in the auditory cortex. FEBS J. 282, 2758–2774. Toda, C., Diano, S., 2014. Mitochondrial UCP2 in the central regulation of metabolism. Best Pract. Res. Clin. Endocrinol. Metabol. 28, 757–764. Verstreken, P., Ly, C.V., Venken, K.J., Koh, T.W., Zhou, Y., Bellen, H.J., 2005. Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47, 365–378. Zeng, L., Yang, Y., Hu, Y., Sun, Y., Du, Z., Xie, Z., Zhou, T., Kong, W., 2014. 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CRediT authorship contribution statement Zheng-De Du: Formal analysis, Writing - original draft. Shu-Guang Han: Investigation. Teng-Fei Qu: Investigation. Bin Guo: Investigation. Shu-Kui Yu: Investigation. Wei Wei: Writing - original draft, Investigation. Shuai Feng: Writing - original draft, Investigation. Ke Liu: Formal analysis, Writing - original draft, Writing - review & editing. Shu-Sheng Gong: Formal analysis, Writing - original draft, Writing - review & editing. Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Nos. 81830030, 81700917 and 81771016). References Bedard, K., Krause, K.H., 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313. Bokoch, G.M., Knaus, U.G., 2003. NADPH oxidases: not just for leukocytes anymore. Trends Biochem. Sci. 28, 502–508. Divakaruni, A.S., Brand, M.D., 2011. The regulation and physiology of mitochondrial proton leak. Physiology (Bethesda) 26, 192–205. Donadelli, M., Dando, I., Fiorini, C., Palmieri, M., 2014. UCP2, a mitochondrial protein regulated at multiple levels. Cell. Mol. Life Sci. 71, 1171–1190. Du, Z., Yang, Q., Zhou, T., Liu, L., Li, S., Chen, S., Gao, C., 2014. D-galactose-induced mitochondrial DNA oxidative damage in the auditory cortex of rats. Mol. Med. Rep. 10, 2861–2867. Du, Z., Yang, Y., Hu, Y., Sun, Y., Zhang, S., Peng, W., Zhong, Y., Huang, X., Kong, W., 2012. A long-term high-fat diet increases oxidative stress, mitochondrial damage and apoptosis in the inner ear of D-galactose-induced aging rats. Hear. Res. 287, 15–24. Du, Z., Yang, Q., Liu, L., Li, S., Zhao, J., Hu, J., Liu, C., Qian, D., Gao, C., 2015. NADPH oxidase 2-dependent oxidative stress, mitochondrial damage and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging rats. Neuroscience 286, 281–292. Du, Z.D., Yu, S., Qi, Y., Qu, T.F., He, L., Wei, W., Liu, K., Gong, S.S., 2019a. NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats. Neurochem. Int. 124, 31–40. Du, Z.D., He, L., Tu, C., Guo, X.A., Yu, S., Liu, K., Gong, S., 2019b. Mitochondrial DNA 3,860-bp deletion increases with aging in the auditory nervous system of C57BL/6J mice. ORL J. Otorhinolaryngol. Relat. Spec. 1–9. Glowatzki, E., Fuchs, P.A., 2002. Transmitter release at the hair cell ribbon synapse. Nat. Neurosci. 5, 147–154. Griesinger, C.B., Richards, C.D., Ashmore, J.F., 2005. Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse. Nature 435, 212–215. Han, C., Linser, P., Park, H.J., Kim, M.J., White, K., Vann, J.M., Ding, D., Prolla, T.A., Someya, S., 2016. Sirt1 deficiency protects cochlear cells and delays the early onset of age-related hearing loss in C57BL/6 mice. Neurobiol. Aging 43, 58–71. Hiona, A., Leeuwenburgh, C., 2008. The role of mitochondrial DNA mutations in aging
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Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice
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Zheng-De Dua, Shu-Guang Hana, Teng-Fei Qua, Bin Guoa, Shu-Kui Yua, Wei Weib, Shuai Fengc, Ke Liua,∗∗, Shu-Sheng Gonga,∗ a
Department of Otorhinolaryngology, Beijing Friendship Hospital, Capital Medical University, 95 Yongan Road, Xicheng District, Beijing, 100050, China Department of Otology, Shengjing Hospital, China Medical University, 36 Sanhao Street, Heping District, Shenyang, 110004, China c Department of Otorhinolaryngology, The First Hospital of China Medical University, 155 Nanjingbei Street, Heping District, Shenyang, 110000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Presbycusis Cochlear ribbon synapsis Mitochondria Oxidative damage D-galactose
Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.
1. Introduction Presbycusis, also known as age-related hearing loss, results from age-related degeneration of the cochlea and the central auditory system (Howarth and Shone, 2006). Since physiological aging processes in animals and humans develop very slowly and the genetic and environmental backgrounds of humans are heterogeneous, an investigation of presbycusis in healthy organisms is very limited. The chronic administration of D-galactose (D-gal) can rapidly induce aging in rodents. Rodents treated with D-gal exhibit increased oxidative damages, declined mitochondrial functions, weakened immune responses, and attenuated learning and memory (Kumar et al., 2009; Lu et al., 2010;
∗
Rehman et al., 2017). These symptoms reflect those in physiological aging well. Therefore, D-gal-induced aging in rodents is commonly used in research as a model of aging and age-related diseases. In exposed to D-gal-induced aging, a previous study reported that only a small number of marginal cells in the stria vascularis of the cochlea (Du et al., 2012), as well as very few neurons in the central auditory system (Du et al., 2015; Du et al., 2019a; Sun et al., 2015; Zeng et al., 2014), are lost because of the activation of mitochondria-dependent apoptosis. However, research rarely systematically reports the changes of hair cells, spiral ganglion cells (SGCs), and ribbon synapses between the inner hair cells (IHCs) and SGCs in the cochlea of rodents with D-galinduced aging.
Corresponding author. Corresponding author. E-mail addresses: [email protected] (K. Liu), [email protected] (S.-S. Gong).
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https://doi.org/10.1016/j.neuint.2019.104649 Received 20 October 2019; Received in revised form 15 December 2019; Accepted 19 December 2019 Available online 20 December 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved.
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10% solution of sodium EDTA for 3 d at 4 °C. The cochleae of one side were further treated for cochlear frozen sections; those of the contralateral side were used for cochlear surface preparations. After decalcification, the cochleae used for frozen sections were incubated overnight at 4 °C in 100 mM sodium phosphate buffer containing 30% sucrose and placed afterwards in optimal cutting temperature-embedding compound (Leica Microsystems, Germany). Sections (10 μm) from cryofixed tissues were collected on 3-aminopropyl-trimethoxysilanecoated slides (Sigma-Aldrich) and dried for 2 h in preparation for the H &E staining or immunohistochemical analysis. The cochlear sections stained with H&E were examined under a light microscope (Nikon, Tokyo, Japan). The Rosenthal's canal was divided into three regions: apex, middle, and base. In each mouse, we evaluated every third modiolar section obtained from 1 cochlea for a total of 10 sections. Tissues from the same animals were used for SGC counting.
In this study, we used the D-gal-induced aging model in mice to determine changes in the numbers of outer hair cells (OHCs), IHCs, SGCs, and ribbon synapses and to observe its influence on the morphology of hair cell stereocilia. We also measured the levels of oxidative stress, oxidative damage to mitochondrial DNA, mitochondrial DNA mutation, and ATP production in the cochlea. The present study may further uncover D-gal-induced morphological changes in the cochlea and potential causative mechanisms of presbycusis. 2. Material and methods 2.1. Animals and D-gal administration Sixty 5-week-old male C57BL/6J mice were obtained from the Experimental Animal Centre of Capital Medical University. The mice were housed in a temperature-controlled (20–22 °C) room and had free access to food and drinking water. After acclimation for a week, the mice were randomly divided into three groups (n = 20 per group) depending on the administered D-gal (Sigma-Aldrich, USA) dose as follows: Control group, the mice were injected subcutaneously with 0.9% saline (the vehicle of D-gal) once a day for 6 weeks; D-gal-L (lowdose D-gal) group, the mice were injected subcutaneously with 500 mg/ kg D-gal once a day for 6 weeks; and D-gal-H (high-dose D-gal) group, the mice were injected subcutaneously with 1000 mg/kg D-gal once a day for 6 weeks. After the measurement of auditory functions, the mice were killed, and both sides of the cochleae were dissected for the determination of the H2O2 content, total superoxide dismutase (T-SOD) activity, and ATP levels, as well as for the extraction of genomic DNA, total RNA, and protein. Alternatively, the cochleae of one side were perfused with 4% paraformaldehyde for cochlear sections and surface preparations, whereas the cochleae of the contralateral side were perfused with 2.5% glutaraldehyde for scanning electron microscopy. All protocols conformed to the Guideline for the Care and Use of Laboratory Animals of the National Institutes of Health. The study protocols were approved by the Committee on the Ethics of Animal Experiments of Capital Medical University.
2.4. Immunohistochemistry of cochlear surface preparations The numbers of OHCs, IHCs, and ribbon synapses were counted using the cochlear surface preparation (n = 4 per group) technique (Han et al., 2016; Kujawa and Liberman, 2009). After decalcification, the softened otic capsule, stria vascularis, tectorial membrane, and Reissner's membrane were removed under an anatomy microscope. The remaining cochlear sensory epithelium was permeabilized in 0.3% Triton X-100 solution for 1 h at room temperature. The specimens were then washed three times with phosphate-buffered saline and blocked with 10% goat serum for 1 h at room temperature. The specimens were incubated with monoclonal mouse anti-carboxyl-terminal binding protein 2 (CtBP2) IgG1 (diluted 1:400; BD Biosciences, USA) and polyclonal rabbit anti-myosin VIIa IgG (diluted 1:400; Abcam, USA) primary antibodies overnight at 4 °C. After washing three times, the specimens were incubated with Alexa Fluor 568- and Alexa Fluor 594conjugated secondary antibodies at a concentration of 1:300 at room temperature for 2 h in darkness. After a final wash, the cochlear sensory epithelia were divided into three segments (apical, middle, and basal turn) and mounted on slides with mounting medium containing 4′,6diamidino-2-phenylindole (DAPI; ZSGB-BIO, China). DAPI-labeled OHCs, myosin VIIa-labeled IHCs, and CtBP2-labeled synaptic ribbons were then examined with a laser scanning confocal microscope (TCS SP8; Leica, Germany). Survival rates of OHCs and IHCs were quantitively evaluated by counting residual hair cells in the cochlea. Synaptic ribbons were normalized to the number of IHCs in the cochlea.
2.2. Auditory function evaluation The auditory function of mice was evaluated by auditory brainstem response (ABR) (Someya et al., 2010). A total of 18 mice (n = 6 per group) were anesthetized by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture. The sound delivery tube of an inserted earphone was tightly fitted into the external auditory canal; subdermal needle electrodes were inserted at the vertex (active), right mastoid (reference), and left mastoid (ground). ABR responses were measured with a tone burst stimulus (3 ms duration, 1 ms rise/fall times, at a rate of 21.1/s) at 8 kHz (low frequency), 16 kHz (middle frequency), and 32 kHz (high frequency) using the TDT System 3 (Tucker-Davis Technologies, Alachua, FL, USA) starting from a 90-dB sound pressure level (SPL) and decreasing in 5-dB increments to the acoustic threshold. At each frequency, determine the ABR threshold, which refers to the minimal SPL resulting in a reliable ABR recording with one or more distinguishable waves that can be clearly identified by visual inspection. It is necessary to repeat the process for low SPLs around the threshold to ensure the consistency of the waveforms. The threshold was independently determined by two researchers who were blinded to the group assignment.
2.5. Immunohistochemistry of cochlear frozen sections The oxidative damage to mitochondrial DNA was evaluated by 8hydroxy-2-deoxyguanosine (8-OHdG) immunohistochemical analysis (Du et al., 2019a). The cochlear sections were washed and incubated with 0.3% Triton X-100 solution for 30 min at room temperature. They were washed again and blocked with 10% goat serum for 1 h at room temperature. Next, slides were incubated with monoclonal mouse anti8-OHdG IgG2b (diluted 1:200; Abcam), monoclonal mouse anti-β-tubulin III IgG2a (diluted 1:100; Abcam), and polyclonal rabbit antimyosin VIIa IgG (diluted 1:400; Abcam) primary antibodies overnight at 4 °C. Slides were then washed and incubated with Alexa Fluor 568-, Alexa Fluor 488-, and Alexa Fluor 594-conjugated secondary antibodies at a concentration of 1:300 at room temperature for 2 h in darkness. After a final wash, sections were mounted with mounting medium containing DAPI (ZSGB-BIO). DAPI-labeled nuclei, 8-OHdG-labeled DNA damage, myosin VIIa-labeled hair cells, and β-tubulin III-labeled SGCs and neurofilaments were captured using a laser scanning confocal microscope (Leica TCS SP8). The expression of 8-OHdG was analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., USA). In the negative control, sections were treated in the same manner, except that incubation with primary antibody was omitted.
2.3. Hematoxylin and eosin (H&E) staining The general morphology of the cochlea and the number of SGCs were analyzed by H&E staining (Someya et al., 2009). Twelve mice (n = 4 per group) were decapitated, and the cochleae of both sides were removed from the temporal bones and fixed in 4% paraformaldehyde overnight at 4 °C. The cochleae were then decalcified in a 2
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used to amplify the mtDNA 3860-bp deletion and 12S rRNA were previously described (Zhang et al., 2009, 2013) and as follows: 3860-bp deletion: forward, 5′-TCATTCTAGCCTCGTACCAACA-3′, reverse, 5′-GAGGTCTGGGTCATTTTCGTTA-3’; 12S rRNA: forward, 5′-ACCGCG GTCATACGATTAAC-3′, reverse, 5′-CCCAGTTTGGGTCTTAGCTG-3’. PCR amplification was performed on a StepOne Real-Time PCR System (Applied Biosystems, USA) in a 25-μl reaction volume consisting of 12.5 μl of 2 × SYBR PCR mix (Tiangen Biotech Co.), 1.0 μl each of forward and reverse primers (7.5 μM), 2.5 μl of sample DNA (20 ng/μl), and 8.0 μl of distilled water. The cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The cycle number at which a significant increase in normalized fluorescence was first detected was designated as the threshold cycle number (Ct). The ratio of mtDNA 3860-bp deletion to total mtDNA was calculated as ΔCt = (Ct3860-bp deletion − Ct12S rRNA), and the relative expression was calculated as 2−ΔΔCt. The PCR products of the mtDNA 3860-bp deletion were cloned and verified using an ABI Prism 377XL sequencer (Applied Biosystems, USA).
2.6. Scanning electron microscopy The ultrastructure of hair cell stereocilia was observed using scanning electron microscopy (Marie et al., 2018). Twelve mice (n = 4 per group) were decapitated, and their cochleae were prepared as described for the H&E staining procedure. After exposing the cochlear sensory epithelium, it was dehydrated in a graded series of ethanol, criticalpoint dried in CO2, coated with gold palladium, and observed using a scanning electron microscope (S-4800; Hitachi Science System, Ltd., Japan). 2.7. Protein extraction and western blot analysis The levels of NADPH oxidase 2 (NOX2) and uncoupling protein 2 (UCP2) protein expression were determined by western blot analysis in 12 mice (n = 4 per group). After decapitation, the cochleae were rapidly removed bilaterally from the temporal bones. The soft tissues were then harvested from the cochleae under an anatomy microscope and homogenized in a RIPA Lysis Buffer (Beyotime, China) according to the manufacturer's instructions. Total protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime). Of each protein lysate, 25 μg were separated by 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After incubating the membranes in a blocking solution (5% nonfat dry milk in Tris-buffered saline containing 1% Tween-20 [TBST]) for 1 h at room temperature, they were washed briefly in TBST and incubated with anti-NOX2 (diluted 1:500; Abcam), anti-UCP2 (diluted 1:500; Abcam), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; diluted 1:1000; Cell Signaling Technology, USA) primary antibodies overnight at 4 °C. After three additional washing steps in TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted 1:5000; ZSGB-BIO) for 1 h at room temperature. Finally, the membranes were visualized in a darkened room using BeyoECL Plus (Beyotime). The detected bands were quantified using the Image-Pro Plus 6.0 software. GAPDH was used as an internal control.
2.10. Statistical analysis The data are presented as the mean ± standard deviation (SD). Statistical tests were performed with SPSS 13.0 software (SPSS Inc., USA). Statistical significance was analyzed via independent-samples ttests or one-way ANOVA. The independent-samples t-test was used to evaluate differences between the Control, and D-gal-H groups. The least significant difference (LSD) post hoc-test in ANOVA was used to evaluate differences among the Control, D-gal-L, and D-gal-H groups. Differences with a P-value < 0.05 were considered to be statistically significant. 3. Results 3.1. Auditory impairment induced by D-gal The auditory functions of mice in the Control group and the D-gal groups were evaluated using the ABR test. In mice, ABR waves consist of five vertex-positive peaks, and wave I reflects the function of cochlear ribbon synapses. We found that the ABR waves I-IV of mice exposed to D-gal-induced aging displayed a poorer peak morphology than those of control mice (Fig. 1A). In the Control, D-gal-L, and D-galH groups, the mean ABR thresholds of mice were 27 ± 4.08 dB SPL, 30 ± 10.00 dB SPL, and 31 ± 9.17 dB SPL at 8 kHz (low frequency), respectively; 33 ± 5.16 dB SPL, 36 ± 3.76 dB SPL, and 38 ± 5.24 dB SPL at 16 kHz (middle frequency), respectively; and 52 ± 6.06 dB SPL, 54 ± 6.65 dB SPL, and 58 ± 7.58 dB SPL at 32 kHz (high frequency), respectively. There were no significant differences among the three groups in all frequencies examined (P > 0.05, Fig. 1B). In the Control, D-gal-L, and D-gal-H groups, the mean amplitudes of the ABR wave I at 90 dB SPL were 1.15 ± 0.42 μV, 0.70 ± 0.30 μV, and 0.53 ± 0.18 μV at 8 kHz, respectively; 0.91 ± 0.27 μV, 0.67 ± 0.20 μV, and 0.53 ± 0.13 μV at 16 kHz, respectively; and 0.55 ± 0.09 μV, 0.40 ± 0.11 μV, and 0.31 ± 0.08 μV at 32 kHz, respectively. Compared with control mice, the mean amplitude of the ABR wave I was in D-gal-treated mice significantly decreased in all frequencies (P < 0.05 or P < 0.01, Fig. 1C). At 90 dB SPL, the latency values of the ABR wave I in mice of the Control, D-gal-L, and D-gal-H groups were 1.97 ± 0.09 ms, 2.02 ± 0.15 ms, and 2.23 ± 0.29 ms at 8 kHz, respectively; 1.96 ± 0.10 ms, 2.04 ± 0.17 ms, and 2.06 ± 0.22 ms at 16 kHz, respectively; and 2.16 ± 0.22 ms, 2.47 ± 0.49 ms, and 2.71 ± 0.46 ms at 32 kHz, respectively. Only in mice of the D-gal-H group, the ABR wave I latency was significantly increased at 8 kHz and 32 kHz (both P < 0.05) in comparison to control mice, whereas no significant differences were detected between the Control group and the D-gal-L group (Fig. 1D).
2.8. H2O2, T-SOD activity, and ATP measurements Twelve mice (n = 4 per group) were sacrificed, and the cochleae of both sides were rapidly removed from the temporal bones. The soft tissues were then harvested from the cochleae under an anatomy microscope and homogenized in cold saline. The homogenate was centrifuged at 4000×g for 15 min at 4 °C, and the supernatant was used for H2O2, T-SOD activity, and ATP measurements. Protein concentrations were evaluated using the Enhanced BCA Protein Assay Kit (Beyotime). H2O2, T-SOD activity, and ATP levels in the cochleae of different groups were quantified using colorimetric kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. These results are representative of at least three separate experiments. 2.9. DNA isolation and mitochondrial DNA (mtDNA) 3860-bp deletion assay The accumulation of mtDNA with 3860-bp deletion was evaluated in 12 mice (n = 4 per group) using a real-time PCR assay (Zhang et al., 2009, 2013). After rapid removal of the bilateral cochleae from the temporal bones, the soft tissues of the cochleae were harvested under an anatomy microscope for genomic DNA isolation. The DNA was extracted using a Genomic DNA Isolation kit (Tiangen Biotech Co., China) according to the manufacturer's instructions. The DNA concentration of each specimen was measured with the GeneQuant pro DNA/RNA Calculator (Amersham Pharmacia Biotech, Sweden). Due to the rarity of the deletion, the 12S rRNA gene was used as an internal control for the murine mitochondrial genome (GenBank: NC_006914). The primers 3
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Figure 1. Auditory function parameters in the Control, D-gal-L, and D-gal-H groups. (A) Representative ABR traces in mice of the Control and the D-gal-H groups at 8 kHz & 90 dB SPL. (B) Mean values of the ABR threshold in the Control, D-gal-L, and D-gal-H groups. (C) Mean amplitudes of the ABR wave I in the Control, D-gal-L, and D-gal-H groups at 90 dB SPL. (D) Mean latency values of the ABR wave I in the Control, D-gal-L, and D-gal-H groups at 90 dB SPL. Data are expressed as the mean ± SD of 6 mice per group. *P < 0.05 or **P < 0.01 versus the Control group.
IHCs were rarely lost in both the Control and the D-gal-H group.
3.2. Tissue morphology in the cochlea of mice presenting D-gal-induced aging
3.3. D-gal-induced insult to cochlear ribbon synapses
The general cochlea morphology was observed in H&E-stained cochlear sections. The cochlea contains an apical, middle, and basal turn (Fig. 2A); the organ of Corti contains in every turn one row of IHCs and three rows of OHCs (Fig. 2C). No loss of IHCs, OHCs, or SGCs was discovered in the cochlear sections of the D-gal-H group (Fig. 2B–D). The number of DAPI-labeled OHCs in cochlear surface preparations (Fig. 2E) was not significantly different between the Control and the Dgal-H group (Fig. 2F). Additionally, the stereocilia of IHCs and OHCs were observed using scanning electron microscopy. Although the stereocilia of OHCs in the D-gal-H group showed mild flattening compared with those in the Control group (Fig. 2G), the stereocilia of OHCs and
The numbers of IHCs and ribbon synapses were also counted in cochlear surface preparations. The IHC ribbon, labeled by CtBP2, is the presynaptic structure of ribbon synapses, and it also allows a suitable estimate for the number of auditory nerve fibers (Fig. 3A). The numbers of myosin VIIa-labeled IHCs were not significantly different among the Control, D-gal-L, and D-gal-H groups (P > 0.05; Fig. 3B, D). CtBP2labeled IHC ribbons were visible on the basal side of the IHCs (Fig. 3B). In the Control, D-gal-L, and D-gal-H groups, the mean numbers of synaptic ribbons per IHC were 14.75 ± 0.96, 12.50 ± 0.58, and 10.75 ± 1.50 in the apical turn of the cochlea, respectively; Fig. 2. Cochlear tissue morphology in mice of the Control group and the D-gal-H group. (A) Representative image of a cochlear section from the D-gal-H group. (B) SGCs in the middle turn of a cochlea from the D-gal-H group. (C) The organ of Corti (OC) in the middle turn of a cochlea from the D-gal-H group. (D) Mean numbers of SGCs in the Control group and the D-gal-H group. (E) Representative images of DAPI-labeled OHCs (blue) in the basal turn of a cochlea. (F) OHC survival rates in the Control and D-gal-H groups. (G) Representative images of hair cell stereocilia in the basal turn of a cochlea under a scanning electron microscope. Data are expressed as the mean ± SD of 4 mice per group. Scale bars: A, 500 μm; B, C, E, 50 μm; G, 5 μm.
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Fig. 3. Numbers of IHCs and synaptic ribbons in the Control, D-gal-L, and D-gal-H groups. (A) Schematic of the cochlear ribbon synapses showing an IHC (blue) and its afferent innervation as it appears in the cochlea immunostained for synaptic ribbon protein (CtBP2: red) and auditory nerve fibers (NF: green). (B) Representative cochlear surface preparation images of CtBP2-labeled synaptic ribbons (red) and myosin VIIa-labeled IHCs (blue) in the basal turn of cochleae from the Control, D-gal-L, and D-gal-H groups. (C) Representative cochlear section images of β-tubulin III-labeled NF (green), myosin VIIa-labeled IHCs (blue), and DAPIlabeled nuclei (purple) in the basal turn of cochleae from the Control, D-gal-L, and D-gal-H groups. (D) IHC survival rate in the apical, middle, and basal turn of cochleae from mice of the Control, D-gal-L, and D-gal-H groups. (E) Synaptic ribbons per IHC in the apical, middle, and basal turn of cochleae from the Control, D-galL, and D-gal-H groups. Data are expressed as the mean ± SD of 4 mice per group. *P < 0.05 or **P < 0.01 versus the Control group, ^P < 0.05 versus the D-gal-L group. Scale bars: B, 50 μm; C, 100 μm. Fig. 4. NOX2-and UCP2-mediated oxidative stress in cochleae of the Control, D-gal-L, and D-gal-H groups. (A) Representative images of NOX2 and UCP2 expression in cochleae of the Control, D-gal-L, and D-gal-H groups using western blotting. (B) Relative protein expression of NOX2 and UCP2 in cochleae of the Control, D-gal-L, and D-gal-H groups. (C) H2O2 levels in cochleae of the Control, D-gal-L, and D-gal-H groups. (D) T-SOD activity levels in cochleae of the Control, D-gal-L, and D-gal-H groups. *P < 0.05 or **P < 0.01 versus the Control group, ^^P < 0.01 versus the D-gal-L group.
middle, and basal turn of the cochlea in comparison to those in the Dgal-L group (P < 0.05, Fig. 3E). To further evaluate the D-gal-induced insult to ribbon synapses, the auditory nerve fibers were examined in immunohistochemical staining using cochlear sections. As shown in Fig. 3C, the positive expression of β-tubulin III was in the SGCs, auditory nerve fibers between SGCs and IHC, and non-neuronal cells lining the inner sulcus. Although the morphological characteristics of β-
16.25 ± 0.96, 12.50 ± 1.29, and 10.50 ± 1.29 in the middle turn, respectively; and 13.50 ± 1.29, 11.25 ± 0.96, and 8.25 ± 1.71 in the basal turn, respectively. Compared with the Control group, the synaptic ribbons per IHC of the D-gal-L group exhibited a statistically significant decrease in the apical, middle, and basal turn of the cochlea (P < 0.05 or P < 0.01, Fig. 3E). Similarly, the numbers of synaptic ribbons in the D-gal-H group were significantly reduced in the apical, 5
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tubulin III-labeled nerve fibers did not allow an accurate quantification of fibers per IHC, the decreased density of β-tubulin III-positive elements in both the D-gal-L and the D-gal-H group was obvious in qualitative assessments (Fig. 3C).
IHCs and SGCs induced by D-gal. Compared with the Control group, the expression of 8-OHdG in IHCs of the D-gal-L and the D-gal-H group was significantly increased by the factor of 3.33 ± 0.31 and 9.65 ± 1.54, respectively (both P < 0.01, Fig. 5A and B), whereas the 8-OHdG expression in SGCs of the D-gal-L group and the D-gal-H group was increased 2.10 ± 0.38-fold and 2.77 ± 0.42-fold, respectively (both P < 0.01; Fig. 5A, C).
3.4. Oxidative stress in the cochlea of mice exposed to D-gal-induced aging The NADPH oxidase system and mitochondria can generate reactive oxygen species (ROS) (Bedard and Krause, 2007; Hiona and Leeuwenburgh, 2008). Therefore, the protein levels of NOX2 and UCP2 were evaluated using western blot analyses. Compared with the Control group, NOX2 protein levels in the D-gal-L and D-gal-H groups were 1.83 ± 0.16-fold and 4.70 ± 0.64-fold (P < 0.05 and P < 0.01, respectively) increased, whereas UCP2 protein levels in those groups were significantly increased by a factor of 1.64 ± 0.21 and 2.68 ± 0.35, respectively (both P < 0.01; Fig. 4A and B). To further investigate the oxidative stress in the cochlea of D-gal-treated mice, we measured the levels of H2O2 and T-SOD activity. H2O2 levels in the Control, D-gal-L, and D-gal-H groups were 14.91 ± 1.07 mmol/g protein, 24.52 ± 3.60 mmol/g protein, and 34.50 ± 1.66 mmol/g protein, respectively. The statistical analysis revealed that the H2O2 levels in the D-gal-L group and in the D-gal-H group were significantly higher than that in the Control group (both P < 0.01, Fig. 4C). Moreover, the T-SOD activity levels in the Control, D-gal-L, and D-gal-H groups were 128.00 ± 6.10 U/mg protein, 94.13 ± 4.59 U/mg protein, and 76.78 ± 7.91 U/mg protein, respectively. Similar to the results in the H2O2 measurements, the T-SOD activity levels in both the D-gal-L group and the D-gal-H group were significantly decreased in comparison to the Control group (both P < 0.01, Fig. 4D).
3.6. Accumulation of the mtDNA 3860-bp deletion and decline in ATP levels in the cochlea of D-gal-treated mice The levels of mtDNA mutation in the cochleae of mice exposed to Dgal-induced aging were evaluated by detecting the mtDNA 3860-bp deletion, also known as the age-related common deletion (CD) (Du et al., 2019b). Using real-time PCR, the relative CD levels in the cochleae of the D-gal-L group and the D-gal-H group were significantly increased by the factor of 4.69 ± 1.13 and 8.86 ± 2.69, respectively, in comparison to that of the Control group (P < 0.05 and P < 0.01, respectively; Fig. 6A). The PCR product sequences identified two putative breakage/fusion sites between the two 15-bp repeat (AGCCCTA CTAATTAC) (at 9089–9103 or 12963–12978) and confirmed that the deleted region was 3860 bp long (Fig. 6C). To further evaluate mitochondrial functions, we measured the levels of ATP production using a colorimetric kit. The levels of ATP production in the Control, D-gal-L, and D-gal-H groups were 12.65 ± 0.50 nmol/mg protein, 9.47 ± 0.61 nmol/mg protein, and 7.38 ± 0.77 nmol/mg protein, respectively. The ATP levels in the D-gal-L and D-gal-H groups were significantly decreased compared to those in the Control group (Fig. 6B). 4. Discussion
3.5. Oxidative damage to mtDNA in the murine cochlea after D-gal-induced aging
The current study is the first demonstrating that the cochlear IHC ribbon synapses are the main insult site at early stages of D-gal-induced aging in mice, although marginal cell apoptosis in the stria vascularis of the cochlea has been previously described by Du et al. (Du et al., 2012). Ribbon synapses between IHCs and SGCs are formed as the first afferent
As shown in Fig. 5A, the expression of 8-OHdG, a marker of oxidative DNA damage (Prabhulkar and Li, 2010), was mainly localized in the cytoplasm of IHCs and SGCs of the D-gal-L and D-gal-H groups, indicating an increased oxidative DNA damage in the mitochondria of
Figure 5. 8-OHdG expression in IHCs and SGCs of cochleae from mice of the Control, D-gal-L, and Dgal-H groups. (A) Representative images of 8-OHdG (red) in myosin VIIa-labeled IHCs (green) and β-tubulin III-labeled SGCs (green) of the Control, D-gal-L, and D-gal-H groups. (B) Relative 8-OHdG expression in IHCs of the Control group, the D-gal-L group, and the D-gal-H group. (C) Relative 8-OHdG expression in SGCs of the Control, D-gal-L, and D-gal-H groups. Data are expressed as the mean ± SD of 4 mice per group. **P < 0.01 versus the Control group, ^P < 0.05 or ^^P < 0.01 versus the D-gal-L group. Scale bar: 50 μm.
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Fig. 6. CD accumulation and decline in ATP levels in cochleae of the Control, D-gal-L, and D-gal-H groups. (A) The relative amount of CD in cochleae of the Control, D-gal-L, and D-gal-H groups. (B) Levels of ATP production in cochleae of the Control group, the D-gal-L group, and the D-gal-H group. (C) Validation of CD by sequencing. The arrows point to the putative breakpoint sites between the two underlined 15-bp direct repeat sequence 5′-AGCCCTA CTAATTAC-3' (at 9089–9103 or 12963–12978). Data are expressed as the mean ± SD of 4 mice per group. *P < 0.05 or **P < 0.01 versus the Control group, ^^P < 0.01 versus the D-gal-L group.
in D-gal-treated mice, the density of auditory nerve fibers from SGCs was largely decreased, and this was accompanied by increased mitochondrial oxidative damage in SGCs, suggesting that mitochondria in SGCs have an impact on the auditory nerve fibers from these cells. Taken together, the oxidative damage to mitochondrial DNA and the subsequent dysfunctions in IHCs and SGCs might cause age-related insults to pre- and postsynaptic structures of the cochlear ribbon synapse, respectively. According to the mitochondrial aging theory (Hiona and Leeuwenburgh, 2008), mitochondrial DNA is highly susceptible to oxidative damage during aging because of excess ROS production, the decline in antioxidants, and the lack of protective histones. Du et al. (Du et al., 2012). demonstrated that the NADPH oxidase system is the main source of ROS production in the cochlea of rats exposed to D-gal-induced aging. NOX2, also known as gp91phox, is widely distributed in different cells and tissues (Bokoch and Knaus, 2003), including the cochlea (Kim et al., 2010). NOX2 constitutes with its corresponding subunits a functional enzyme complex that transfers electrons from NADPH to oxygen to generate ROS(Bedard and Krause, 2007). In the current study, we also found NOX2 protein overexpressed in the cochlea of D-gal-treated mice. This indicates that NOX2-generated ROS might be, at least partly, responsible for D-gal-induced mitochondrial DNA damage in IHCs and SGCs. Apart from the NADPH oxidase system, mitochondria are another important source of ROS production in vivo (Hiona and Leeuwenburgh, 2008). Mitochondrial ROS generation can activate UCP2, which is located in the inner mitochondrial membrane (Divakaruni and Brand, 2011; Donadelli et al., 2014; Toda and Diano, 2014). Therefore, the UCP2 expression can indirectly reflect cellular ROS levels. Previous studies demonstrated UCP2 overexpression in the cochlea and the central auditory system of rats exposed to D-gal-induced aging (Du et al., 2014, 2012). In the present study, we also found that the levels of UCP2 protein were significantly increased in the cochlea of D-gal-treated mice, suggesting that mitochondrial ROS may also be involved in the D-gal-induced damage to mitochondrial DNA in IHCs and SGCs.
neuronal connection in the auditory nervous system (Glowatzki and Fuchs, 2002; Meyer et al., 2009). A previous study also found significant synaptic losses long before a decline in ABR threshold values or hair cell counts were detected in early aging stages of CBA/CaJ mice (Sergeyenko et al., 2013). ABR thresholds have been shown to be insensitive to widespread loss of cochlear IHCs ribbon synapses that can occur after noise exposure or at the early stage of aging processes, while the amplitudes of the ABR wave I, which reflect the function of ribbon synapses, were largely decreased (Kujawa and Liberman, 2009; Sergeyenko et al., 2013). Our findings demonstrate that at early stages of D-gal-induced aging, the ABR threshold in mice is not significantly different, but the amplitude and latency of the ABR wave I are significantly decreased and increased, respectively. This indicates that the impairment of the ABR wave I is correlated with synaptic losses. These results further demonstrate that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis. The cochlear ribbon synapse is characterized by a high rate of neurotransmitter release (Griesinger et al., 2005). The effective neurotransmitter transport system in IHCs for the maintenance of synaptic vesicles recycling and tonic exocytosis is largely dependent on ATP produced by mitochondria (Kang et al., 2008; Li et al., 2004; Stowers et al., 2002; Verstreken et al., 2005). In this study, we observed increased oxidative damage to mitochondrial DNA in the IHCs, as well as CD in the cochlea of D-gal-treated mice. Furthermore, we also demonstrated a decrease in mitochondrial ATP production in the cochlea of mice exposed to D-gal-induced aging. These outcomes indicate that mitochondrial oxidative damage and subsequent dysfunctions might be responsible for the impairment of presynaptic structures at the cochlear ribbon synapse. Previously published studies suggest that an age-related loss of auditory nerve fibers, especially low-spontaneous rate fibers, might be an important contributor to age-related hearing loss (Liberman, 1982; Schmiedt et al., 1996). Based on an electron-microscopic study using serial sections of the cat cochlea, the low-spontaneous rate fibers are susceptible to injury because of impaired mitochondria (Liberman, 1980). In the present study, we also found that 7
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In conclusion, this study is the first demonstrating that the cochlear ribbon synapse is the primary insult site in the D-gal-induced aging model and that mitochondrial oxidative damage mediated by the NADPH oxidase system and mitochondria might be responsible for this insult. Physiological aging in the cochlea can be experimentally modeled by D-gal administration, and D-gal-induced aging in mice is an ideal animal model to investigate mechanisms and interventional strategies of age-related insults to cochlear ribbon synapses in early stages of presbycusis.
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CRediT authorship contribution statement Zheng-De Du: Formal analysis, Writing - original draft. Shu-Guang Han: Investigation. Teng-Fei Qu: Investigation. Bin Guo: Investigation. Shu-Kui Yu: Investigation. Wei Wei: Writing - original draft, Investigation. Shuai Feng: Writing - original draft, Investigation. Ke Liu: Formal analysis, Writing - original draft, Writing - review & editing. Shu-Sheng Gong: Formal analysis, Writing - original draft, Writing - review & editing. Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Nos. 81830030, 81700917 and 81771016). References Bedard, K., Krause, K.H., 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313. Bokoch, G.M., Knaus, U.G., 2003. NADPH oxidases: not just for leukocytes anymore. Trends Biochem. Sci. 28, 502–508. Divakaruni, A.S., Brand, M.D., 2011. The regulation and physiology of mitochondrial proton leak. Physiology (Bethesda) 26, 192–205. Donadelli, M., Dando, I., Fiorini, C., Palmieri, M., 2014. UCP2, a mitochondrial protein regulated at multiple levels. Cell. Mol. Life Sci. 71, 1171–1190. Du, Z., Yang, Q., Zhou, T., Liu, L., Li, S., Chen, S., Gao, C., 2014. D-galactose-induced mitochondrial DNA oxidative damage in the auditory cortex of rats. Mol. Med. Rep. 10, 2861–2867. Du, Z., Yang, Y., Hu, Y., Sun, Y., Zhang, S., Peng, W., Zhong, Y., Huang, X., Kong, W., 2012. A long-term high-fat diet increases oxidative stress, mitochondrial damage and apoptosis in the inner ear of D-galactose-induced aging rats. Hear. Res. 287, 15–24. Du, Z., Yang, Q., Liu, L., Li, S., Zhao, J., Hu, J., Liu, C., Qian, D., Gao, C., 2015. NADPH oxidase 2-dependent oxidative stress, mitochondrial damage and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging rats. Neuroscience 286, 281–292. Du, Z.D., Yu, S., Qi, Y., Qu, T.F., He, L., Wei, W., Liu, K., Gong, S.S., 2019a. NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats. Neurochem. Int. 124, 31–40. Du, Z.D., He, L., Tu, C., Guo, X.A., Yu, S., Liu, K., Gong, S., 2019b. Mitochondrial DNA 3,860-bp deletion increases with aging in the auditory nervous system of C57BL/6J mice. ORL J. Otorhinolaryngol. Relat. Spec. 1–9. Glowatzki, E., Fuchs, P.A., 2002. Transmitter release at the hair cell ribbon synapse. Nat. Neurosci. 5, 147–154. Griesinger, C.B., Richards, C.D., Ashmore, J.F., 2005. Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse. Nature 435, 212–215. Han, C., Linser, P., Park, H.J., Kim, M.J., White, K., Vann, J.M., Ding, D., Prolla, T.A., Someya, S., 2016. Sirt1 deficiency protects cochlear cells and delays the early onset of age-related hearing loss in C57BL/6 mice. Neurobiol. Aging 43, 58–71. Hiona, A., Leeuwenburgh, C., 2008. The role of mitochondrial DNA mutations in aging
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