1. Introduction
Approximately one in five people over the age of 50 has imperfect hearing, and almost half of those aged over 65 years have hearing difficulties [
1,
2,
3,
4]. Presbycusis, also termed age-related hearing loss (ARHL), is an irreversible hearing impairment associated with aging due to limited repair capacity of sensorineural tissues in the cochlea. Unfortunately, there is no effective cure for the patients, and future treatment development is still questionable due to lack of mechanistic insight [
5]. In order to identify pathologic changes in the aged cochleae, we utilized C57BL/6J mice to investigate the pathophysiology of ARHL, as this strain displays accelerated, high-frequency hearing loss by 3–6 months of age and profound hearing impairment by 15 months of age [
6,
7,
8,
9,
10,
11].
Mitochondria are involved in the metabolic dysregulation associated with ARHL pathology. Morphologically, damage is apparent in the outer hair cells (OHCs) of animal ARHL models [
10,
12,
13]. Mitochondria are the principal source of reactive oxygen species (ROS), the production of which is closely associated with ARHL progression. Antioxidants alleviate the deleterious effects of ROS and effectively treat oxidative stress-related diseases in an animal model of ARHL [
14]. As mitochondria play key roles in both the respiratory chain and cell death, animal models of ARHL often exhibit defects in mitochondrial enzyme activities and mitochondrial-mediated apoptosis. Idh2-knockout mice exhibit accelerated ARHL progression, accompanied by a profound loss of hair cells and spiral ganglion neurons (SGNs), an increase in oxidative damage, and increased apoptotic cell death [
15]. The mitochondrial proapoptotic BCL2-antagonist/killer 1 (Bak) gene mediates ARHL in C57BL/6J mice by enhancing mitochondrial fission and cellular apoptosis, both of which are systematic responses to oxidative stress [
10].
Neuronal cell death occurs through various pathways in sensorineural tissue, leading to hearing impairment [
2,
5,
10,
16]. Necroptosis is a programmed cell death that exhibits necrosis-like morphological characteristics. Necroptosis is activated by the receptor-interacting protein (RIP) homology interaction motif (RHIM), and is mediated by proteins such as RIP3 and the mixed-lineage kinase domain-like (MLKL) protein [
17]. Unlike apoptosis, necroptosis permeabilizes both intra- and extracellular membranes, releasing cellular and organelle contents into the extracellular medium and inducing inflammation. Necroptosis plays an important role in the pathogenesis of aging, neurodegenerative diseases, and hearing impairments including cisplatin- and aminoglycoside-induced ototoxicity and noise-induced hearing loss [
17,
18]. However, to the best of our knowledge, cochlear necroptosis has not been described in ARHL animal models. As successful modulation of cell death pathways may lead to potential development of clinically applicable drugs, we aimed to investigate the pathophysiology of ARHL by focusing on mitochondrial damage and necroptosis.
2. Materials and Methods
2.1. Experimental Animals and Design
All animal experiments were approved by Chungnam National University, Institutional Animal Care and Use Committee (IACUC, 9 January 2016). All animal care and use was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. C57BL/6J male mice, aged 2 months or 20 months, were used in this study.
2.2. Auditory Brainstem Response (ABR)
ABR thresholds at frequencies between 4 and 32 kHz, and click sounds, were obtained separately from both ears as described previously [
19]. The TDT System-3 (Tucker Davis Technologies, Gainesville, FL, USA) hardware and software were used to obtain the ABRs. The stimuli were computer-generated tone pips. The animals were anesthetized with intramuscular injection of zolazepam HCl 40 mg/kg (Zoletil, Virbac Animal Health, Carros, France) and xylazine 10 mg/kg (Rompun, Bayer Animal Health, Monheim, Germany). Subcutaneous needle electrodes were placed around the skull vertex and both infraauricular areas. Tone bursts, with a duration of 4 ms and rise-fall time of 1 ms at frequencies of 4, 8, 16, and 32 kHz, were used, in addition to clicks. The sound intensity was varied in 10 dB increments for the tone burst sounds and in 5 dB increments for the click and tone burst sounds close to the threshold. The contralateral ear was not masked because the stimuli were transmitted through a sealed earphone. The waveforms were analyzed using a custom program (BioSig RP, ver. 4.4.1; Tucker Davis Technologies) with the researcher blinded to the treatment group. Threshold was defined as the lowest stimulus intensity to evoke a wave III response > 0.2 μV.
2.3. Tissue Preparation and Immunofluorescence
Samples from mice aged 2 and 20 months were collected. Cochlear tissues were obtained to localize inner and outer hair cells and neuronal fibers as previously described [
20,
21]. Briefly, tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at room temperature to remove the cochlear bony walls and lateral wall tissues and separated individual cochlear turns. The remaining cochlear tissues were prepared within immunofluorescence. Tissues were blocked in 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in 5% normal goat serum (Vector Laboratories, Burlingame, CA) for 1 h, then incubated with rabbit anti-myosin VIIa primary antibody (Proteus BioSciences, Ramona, CA)—Alexa Fluor 488 Phalloidin (A12379; Invitrogen-Molecular Probes, Eugene, OR); rabbit anti-CtBP2 primary antibody (BD Biosciences)—Alexa Fluor 594 Phalloidin (A11034; Invitrogen-Molecular Probes, Eugene, OR); chicken anti-NF-H primary antibody (Millipore)—Alexa Fluor 488 Phalloidin (A11039; Invitrogen-Molecular Probes, Eugene, OR); mouse anti-COX1 (also known as MTCO1) primary antibody (Invitrogen); rabbit anti-RIPK3 primary antibody (Novus Biologicals); or Hoechst33342 (H3570; Invitrogen-Molecular Probes, Eugene, OR) at a concentration of 1:200 in blocking solution overnight at 4 °C (1:1000 for Hoechst33342 for 1 min). After rinsing six times in PBS for 10 min, the tissues were incubated with each secondary antibody at a concentration of 1:200 in PBS for 2 h. The specimens were mounted on glass slides using Crystalmount (Biomeda, Foster City, CA) and observed under an epifluorescence microscope (Zeiss Axio Scope A1; Zeiss, Oberkochen, Germany) with a digital camera.
2.4. Transmission Electron Microscopy (TEM)
The decalcificated mouse cochlea were prefixed immediately in 2.5% glutaraldehyde–2% paraformaldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) for 2 h at 4 °C. After washing with sodium cacodylate buffer, tissue samples were postfixed in 2% osmium tetroxide–1.5% ferrocyanide in 0.15M cacodylate buffer (pH 7.4) for 1 h. Then, samples were incubated with 1% TCH for 30 min and treated with 2% OsO4 for 30 min. Subsequently, samples were En bloc stained with 1% uranyl acetate overnight at 4 °C and lead citrate for 30 min at 60 °C. The tissues were then embedded in Epon 812 mixture after dehydration in an ethanol and propylene oxide series. Polymerization was conducted with pure resin at 70 °C for 24 h. Sections (200 nm) were obtained with an ultramicrotome (Ultra Cut-UCT, Leica, Vienna, Austria) and then collected on 100 mesh copper grids. The sections were visualized using conventional TEM (JEM-1400Plus) at 120 kV and Bio-HVEM (JEM-1000BEF, JEOL, Tokyo, Japan) at 1000 kV. The sections were visualized using Bio-HVEM system (JEM-1400Plus at 120 kV and JEM-1000BEF at 1000 kV, JEOL, JAPAN).
2.5. Measurement of Cochlear Blood Flow
The left tympanic bulla of each mouse was exposed and opened under anesthesia. After the mouse was placed on the stereotaxic instrument, the cochlear blood flow was measured using a 0.1 mm diameter laser Doppler probe placed over the lateral wall of the cochlea. Cochlear blood flow was determined from an intensity oscillation that was translated from the frequency of the oscillation produced by the Doppler frequency shift of the red blood cells in the left tympanic bulla, using a Laser Doppler Flowmeter (Transonic Systems, Ithaca, NY, USA). Each intensity oscillation was measured separately, and relative cochlear blood flow was reported as the ratio of the control (pre) value to the postnoise exposure value.
2.6. Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Quantitative RT-PCR was performed as previously described [
19,
22]. Briefly, tissues were collected and frozen immediately in liquid nitrogen and homogenized. Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA USA) according to the manufacturer’s protocol. RNA was quantified using a Nano drop (Nano Drop Technologies, Wilmington, DE). cDNA was produced using the cDNA synthesis kit (Roche, Branchburg, NJ, USA). Real time PCR was performed on a CFX Connect Real-Time PCR Detection System (BioRad, Des Plaines, IL, USA) by using a reaction mixture with SYBR Green as the fluorescent dye (Applied Biosystems, Waltham, Massachusetts, USA), a 1/10 vol of the cDNA preparation as template, and 250 nM of each primer (Realtime primers, PA). The fold change in the target gene relative to endogenous control gene (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) was determined by: fold changeXX = 2
−Δ(ΔCT) where ΔC
T = C
T,target gene − C
T,GAPDH and Δ(ΔC
T) = ΔC
T,Aged cochlea − ΔC
T,Young cochlea [
23].
2.7. Image Processing and Statistical Analysis
Adobe Photoshop CS6 was used for adjustment of image contrast, superimposition of images, and colorization of monochrome fluorescence images. An unpaired Student’s t-test was used for all comparisons. A p-value < 0.05 was significant in each case. All tests were performed using GraphPad Prism 6.
4. Discussion
We identified increased RIPK3 level in the aging cochlea, especially in the inner and outer hair cells and stria vascularis. Pronounced reduction in COX1 correlated with the degree of mitochondrial morphological damage and hearing impairment found in aging animals was associated with a loss of sensory hair cells and neuronal filaments. Our data suggest that mitochondrial degeneration and necroptosis may play a critical role in the pathophysiology of ARHL and provide mechanistic insights for future therapeutic development.
Laboratory animals are useful when investigating ARHL because of their short lifespans and well-defined genetics. Like humans, many inbred mouse strains show variable extents of ARHL; the age of onset ranges from 3 months in DBA/2J mice to over 20 months in CBA/CaJ mice [
27,
33,
34,
35]. The C57BL/6J and CBA/CaJ strains are the inbred strains most widely used in hearing research [
33]. The C57BL/6J strain has been extensively employed as a model of early-onset ARHL; the mice exhibit high-frequency hearing loss by 3–6 months and profound hearing impairment by 15 months [
6,
7,
8,
9]. In contrast, CBA/CaJ mice exhibit normal hearing to 15 months or more, and are often used as positive controls [
6,
34,
36]. One well-documented genetic factor responsible for hearing loss in C57BL/6J mice is the recessive
ahl allele of
Cdh23, which encodes cadherin 23 [
37]. However, interestingly, inbred strain variants of
Cdh23 show differences in ARHL onset and progression: the CBA/CaJ-derived
Cdh23Ahl+ allele dramatically reduces hair cell death and hearing loss in a C57BL/6J genetic background, but the C57BL/6J-derived
Cdh23ahl allele has little effect on hearing loss in a CBA/CaJ background [
33].
Cdh23ahl homozygosity is necessary but not sufficient to trigger accelerated hearing loss in C57BL/6J mice [
9,
33]. An interesting study by Frisina et al. showed that F1 (CBA × C57) hybrids exhibit better hearing (“golden ears”) than either parental strain [
38]. Despite the accelerated hearing loss of the C57BL/6J strain, such mice are valuable when studying the features of presbycusis; we used these mice in our current and earlier studies [
10,
11,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48]. The early-onset hearing loss of C57BL/6J mice endures for the lifespan, fitting well with the reality of human hearing loss. The World Health Organization estimated that ~466 million people worldwide, including 34 million children, have some degree of hearing loss.
Necroptosis triggers inflammation and cell death caused by cell lysis. Increasing evidence suggests that necroptosis plays a critical role in the pathogenesis of several neurodegenerative diseases and manifestations of hearing impairment, including cisplatin- and aminoglycoside-induced ototoxicity and noise-induced hearing loss [
17,
18,
49,
50,
51]. Inhibition of necroptosis has been reported to confer neuroprotective effects in animal models of neurodegenerative disorders, and necroptotic factors may thus be promising therapeutic targets [
17]. We provide the first evidence that the aging cochlea exhibits necroptosis in vivo. Few other studies have investigated the role of necroptosis in the cochlea; in these studies, models of ototoxicity and noise-induced hearing loss were employed, but not a model of age-related hearing impairment. Necrostatin-1 (Nec-1, an RIPK1 inhibitor) alleviated noise-induced hearing loss in the mouse [
52], protected spiral ganglion neurons, and improved ABR thresholds in rats exposed to ouabain [
53]. Park et al. reported that NecroX, a necroptosis inhibitor, prevented gentamicin-induced HC loss in neonatal mouse explants of the organ of Corti [
54]. Ruhl et al. reported that in vivo, both necroptosis and apoptosis are involved in cisplatin- and aminoglycoside-induced ototoxicity in both sexes, but, ex vivo, only apoptosis contributed to the ototoxicity [
51] evident in Casp8 and Ripk3 knockout models. The authors thus confirmed earlier genetic evidence that Caspase-8-mediated (extrinsic) apoptosis is involved in cisplatin-mediated ototoxicity. We also show that aging cochleae undergoes at least two programmed cell death pathways simultaneously, apoptosis (
Figure S1F–G) and necroptosis. However, the relative contributions of these pathways to ARHL and cochlea aging are still unknown.
Inflammaging (chronic low-grade inflammation) is a hallmark of aging and is a major risk factor for a variety of age-related diseases, including neurodegenerative diseases, cardiovascular disease, and type 2 diabetes. Despite the strong association among inflammation, aging, and age-associated diseases, the molecular mechanisms that contribute to the chronic, low-grade inflammation observed in aging animals remain unknown. Our initial aim was to explore whether aging affects the sensory, neural, and metabolic features of the cochlea through age-related inflammation and necroptotic stress. We found an association between inflammation and necroptosis in the aging cochlea. Significantly increased necroptosis protein, RIPK3, was significantly increased in the organ of Corti and stria vascularis. Recent studies have identified both resident macrophages (CD163-, IBA1-, and CD68-positive cells) and migrated macrophages in the human cochlea [
55,
56]. It is well-known that IL-1 and IL-6 are important modulators of the innate and adaptive immune responses [
57]. Thus, it would be interesting to explore whether resident or infiltrated immune cells are responsible for the increased cytokine levels, and, where the resident macrophages, if present, are located within the cochlea (i.e.; in the lateral wall, auditory nerve, or elsewhere).
The proinflammatory cytokine TNF-α and its receptor, TNFR (TNF-α receptor), play key roles in cell death machineries, including necroptosis and apoptosis [
58]. Chen et al. reported that TNFα-induced necroptosis in murine fibrosarcoma L929 cells resulted in enriched levels of RIP1/RIP3/MLKL in the mitochondrial associated membrane fraction of cells [
59]. Others show that knockdown or inhibition of
Drp1 (dynamin-related protein 1; also known as
DNM1L, dynamin-1-like protein) protects both HeLa and HT-29 cells from TNFα-mediated necroptosis [
60]. Notably, we also observed a significant increase of
Drp1 expression in aging cochleae in our in vivo system (
Figure S1E).
Our study also contains several weaknesses. First, protein expression levels were measured only by IF assays, and not by Western blot (WB) or Elisa. We employed the IF assays to investigate protein expression in subregions of cochlea rather than detecting signals from the whole cochlea. Another weakness is lack of mitochondrial function studies (e.g.; mitochondrial enzymatic activity) to confirm the mitochondrial dysfunction in ARHL. Finally, although we present two main phenomena (mitochondrial damage and necroptosis) in the aging cochlea, we are unable to suggest a direct relation between mitochondria/ROS and necroptosis. Despite the long assumption that ROS and mitochondria are involved with necroptosis, a previous study showed that necroptosis can occur in the absence of mitochondria or ROS [
61]. Further studies are necessary to examine which specific genes and pathways are involved with the necroptotic cell death in ARHL models; and whether the increase in chronic inflammation actually causes cochlear aging and age-related hearing loss.