Expression profiling of cochlear genes uncovers sex-based cellular function in mouse cochleae.

Sex is a pivotal biological factor that significantly impacts tissue homeostasis and disease susceptibility. In the auditory system, sex differences have been observed in cochlear physiology and responses to pathological conditions. However, the underlying molecular mechanisms responsible for these differences remain elusive. The current research explores the differences in gene expression profiles in the cochlea between male and female mice, aiming to understand the functional implication of sex-biased gene expression in each sex. Using RNA-sequencing analysis on cochlear tissues obtained from male and female mice, we identified a significant number of genes exhibiting sex-biased expression differences. While some of these differentially expressed genes are located on sex chromosomes, most are found on autosomal chromosomes. Further bioinformatic analysis revealed that these genes are involved in several key cellular functions. In males, these genes are notably linked to oxidative phosphorylation and RNA synthesis and processing, suggesting their involvement in mitochondrial energy production and regulatory control of gene expression. In contrast, sex-biased genes are associated with mechano-transduction and synaptic transmission within female cochleae. Collectively, our study provides valuable insights into the molecular differences between the sexes and emphasizes the need for future research to uncover their functional implications and relevance to auditory health and disease development.

Heterogeneity in macrophages along the cochlear spiral in mice: insights from SEM and functional analyses.

The susceptibility of sensory cells to pathological conditions differs between the apical and basal regions of the cochlea, and the cochlear immune system may contribute to this location-dependent variability. Our previous study found morphological differences in basilar membrane macrophages between the apical and basal regions of the cochlea. However, the details of this site-dependent difference and its underlying structural and biological basis are not fully understood. In this study, we utilized scanning electron microscopy to examine the ultrastructure of macrophages and their surrounding supporting structures. Additionally, we examined the phagocytic activities of macrophages and the expression of immune molecules in both apical and basal regions of the cochlea. We employed two mouse strains (C57BL/6J and B6.129P-Cx3cr1tm1Litt/J) and evaluated three experimental conditions: young normal (1-4 months), aging (11-19 months), and noise-induced damage (120 dB SPL for 1 h). Using scanning electron microscopy, we revealed location-specific differences in basilar membrane macrophage morphology and surface texture, architecture in mesothelial cell layers, and spatial correlation between macrophages and mesothelial cells in both young and older mice. Observations of macrophage phagocytic activities demonstrated that basal macrophages exhibited greater phagocytic activities in aging and noise-damaged ears. Furthermore, we identified differences in the expression of immune molecules between the apical and basal cochlear tissues of young mice. Finally, our study demonstrated that as the cochlea ages, macrophages in the apical and basal regions undergo a transformation in their morphologies, with apical macrophages acquiring certain basal macrophage features and vice versa. Overall, our findings demonstrate apical and basal differences in macrophage phenotypes and functionality, which are related to distinct immune and structural differences in the macrophage surrounding tissues.

Mild hearing loss in C57BL6/J mice after exposure to antiretroviral compounds during gestation and nursing.

OBJECTIVE: There is evidence of ototoxicity from antiretrovirals (ARVs), and ARV therapy in pregnant/nursing mothers can expose offspring to these compounds. The current work modelled whether exposure to ARVs in utero and during nursing altered the functioning of the auditory system in offspring mice. DESIGN: The females of seven breeding pairs of C57BL6/J mice were given daily doses of ARVs lamivudine and tenofovir disoproxil fumarate by oral gavage during gestation and nursing. Three breeder females were given equivalent volumes of water as controls. At wean age (3 weeks after birth), the offspring mice were tested with auditory brainstem responses (ABRs). At the conclusion of the experiment, the offspring mice's cochleae were examined for hair cell counts. STUDY SAMPLE: Ten breeder female C57BL6/J mice and 69 offspring mice. RESULTS: The offspring mice exposed to ARVs during development showed higher ABR thresholds than the control offspring. No differences were found in supra-threshold ABRs. There was no evidence of missing hair cells. CONCLUSIONS: Hearing impairment may be a possible consequence of exposure to ARVs during gestation and development. Because the threshold differences were not large, if they are occurring in humans, it is unlikely they would be identified in any hearing screening tests.

Nano-Calcium Carbonate Affect the Respiratory and Function Through Inducing Oxidative Stress: A Cross-sectional Study Among Occupational Exposure of Workers and a Further Research for Underlying Mechanisms.

OBJECTIVE: The aim of the study is to investigate whether nano-calcium carbonate (nano-CaCO 3 ) occupational exposure could induce adverse health effects in workers. METHODS: A cross-sectional study was conducted in a nano-CaCO 3 manufacturing plant in China. Then, we have studied the dynamic distribution of nano-CaCO 3 in nude mice and examined the oxidative damage biomarkers of subchronic administrated nano-CaCO 3 on Sprague-Dawley rats. RESULTS: The forced vital capacity (%) and the ratio of FEV1 to FVC is the rate of one second of workers were significantly decreased than unexposed individuals. Dynamic imaging in mice of fluorescence labeled nano-CaCO 3 showed relatively high uptake and slow washout in lung. Similar to population data, the decline in serum glutathione level and elevation in serum MDA were observed in nano-CaCO 3 -infected Sprague-Dawley rats. CONCLUSIONS: We found that nano-CaCO 3 exposure may result in the poor pulmonary function in workers and lead to the changes of oxidative stress indexes.

Galectin-3 protects auditory function in female mice.

Sex differences in the development of sensorineural hearing loss have been recognized in various inner ear disorders, but the molecular basis for such differences is poorly understood. Autosomal genes have been shown to cause sex differences in disease susceptibility, but many genes exerting sex-dependent effects on auditory function remain to be identified. Galectin-3 (Gal-3), a protein encoded by the autosomal gene Lgals3, is a member of the ß-galactoside-binding protein family, and has been linked to multiple biological processes, including immune responses, apoptosis, and cell adhesion. Here, we investigated auditory function and hair cell integrity in Gal-3 knockout (KO, Lgals3-/-) and wild-type (WT, Lgals3+/+) mice from age 1 to 6 months. KO mice show a more rapid age-related increase in ABR thresholds compared to WT mice. Noticeably, the threshold deterioration in female KO mice is significantly greater than in the male KO and WT mice. The ABR threshold elevation manifests over a broad frequency range in female KO mice, whereas the threshold elevations are confined to high frequencies in the male KO and WT mice. Moreover, DPOAE input/output functions reveal a similar pattern of auditory dysfunction, with the female KO mice displaying a significantly greater reduction in DPOAE amplitudes than male KO mice and WT mice of both sexes. Finally, age-related outer hair cell loss is greater for female KO mice compared to male KO mice and WT mice of both sexes. Together, these results indicate that Gal-3 deficiency exacerbates age-related cochlear degeneration and auditory dysfunction in female mice. Our study identifies Gal-3 as a sex-dependent molecule for maintaining female cochlear integrity.

gom1 Mutant Mice as a Model of Otitis Media.

Otitis media (OM) disease is a common cause of hearing loss that is primarily the result of middle ear infection. At present, our understanding of the mechanisms leading to OM is limited due to the lack of animal models of OM with effusion (OME). Here, we report that the mice with genetic otitis media one (gom1) mutants are prone to OM. gom1 Mice were produced by the N-ethyl-N-nitrosourea (ENU) mutagenesis program as an animal model to study OM. These mice demonstrate many common features of OM, such as middle ear effusion and hearing impairment. We revealed that gom1 mice display various signs of middle ear and inner ear dysfunctions, including elevated thresholds of auditory-evoked brainstem response (ABR) and lack of cochlear microphonic responses. Decreased compliance in tympanometry measurements indicates tympanic membrane and ossicular chain malfunction. We confirmed through histological examinations of middle ear structures that 34/34 (100 %) of the mutant mice suffered from severe OME. While individual ears had different levels of effusion and inflammatory cells in the middle ear cavity, all had thickened middle ear mucosa and submucosa compared to control mice (B6). Moreover, the mutant mice displayed cochlear hair cell loss. These observations also suggested the craniofacial abnormalities in the gom1 mouse model. Together, these results indicate that gom1 mice could be valuable for investigating the genetic contribution to the development of middle ear disease.

Time- and frequency-dependent changes in acoustic startle reflex amplitude following cyclodextrin-induced outer and inner cell loss.

The acoustic startle reflex (ASR) amplitude can be enhanced or suppressed by noise-induced hearing loss or age-related hearing loss; however, little is known about how the ASR changes when ototoxic drugs destroy outer hair cells (OHCs) and inner hair cells (IHCs). High doses of 2-hydroxypropyl-beta-cyclodextrin (HPßCD), a cholesterol-lowering drug used to treat Niemann-Pick Type disease type C1, initially destroy OHCs and then the IHCs 6-8 weeks later. Adult rats were treated with doses of HPßCD designed to produce a diversity of hair cell lesions and hearing losses. When HPßCD destroyed OHCs and IHCs in the extreme base of the cochlea and caused minimal high-frequency hearing loss, the ASR amplitudes were enhanced at 4-, 8- and 16 kHz. Enhanced ASR occurred during the first few weeks post-treatment when only OHCs were missing; little change in the ASR occurred 6-8-WK post-treatment. If HPßCD destroyed most OHCs and many IHCs in the basal half of the cochlea, high-frequency thresholds increased ∼50 dB, and ASR amplitudes were reduced ∼50% at 4-, 8- and 16-kHz. The ASR amplitude reduction occurred in the first few weeks post-treatment when the OHCs were degenerating. The ASR was largely abolished when most of the OHCs were missing over the basal two-thirds of the cochlea and a 40-50 dB hearing loss was present at most frequencies. These results indicate that high-doses of HPßCD generally lead to a decline in ASR amplitude as OHCs degenerate; however, ASR amplitudes were enhanced in a few cases when hair cell loss was confined to the extreme base of the cochlea.

Loss of CX3CR1 augments neutrophil infiltration into cochlear tissues after acoustic overstimulation.

The cochlea, the sensory organ for hearing, has a protected immune environment, segregated from the systemic immune system by the blood-labyrinth barrier. Previous studies have revealed that acute acoustic injury causes the infiltration of circulating leukocytes into the cochlea. However, the molecular mechanisms controlling immune cell trafficking are poorly understood. Here, we report the role of CX3CR1 in regulating the entry of neutrophils into the cochlea after acoustic trauma. We employed B6.129P-Cx3cr1tm1Litt /J mice, a transgenic strain that lacks the gene, Cx3cr1, for coding the fractalkine receptor. Our results demonstrate that lack of Cx3cr1 results in the augmentation of neutrophil infiltration into cochlear tissues after exposure to an intense noise of 120 dB SPL for 1 hr. Neutrophil distribution in the cochlea is site specific, and the infiltration level is positively associated with noise intensity. Moreover, neutrophils are short lived and macrophage phagocytosis plays a role in neutrophil clearance, consistent with typical neutrophil dynamics in inflamed non-cochlear tissues. Importantly, our study reveals the potentiation of noise-induced hearing loss and sensory cell loss in Cx3cr1-/- mice. In wild-type control mice (Cx3cr1+/+ ) exposed to the same noise, we also found neutrophils. However, neutrophils were present primarily inside the microvessels of the cochlea, with only a few in the cochlear tissues. Collectively, our data implicate CX3CR1-mediated signaling in controlling neutrophil migration from the circulation into cochlear tissues and provide a better understanding of the impacts of neutrophils on cochlear responses to acoustic injury.

Phenotypic differences in the inner ears of CBA/CaJ and C57BL/6J mice carrying missense and single base pair deletion mutations in the Cdh23 gene.

Different mutations in the cadherin 23 (CDH23) gene in different genetic backgrounds have been linked to either syndromic or nonsyndromic forms of deafness in humans. We previously reported a progressive hearing loss (HL) mouse model, the Cdh23erl/erl mouse, which carries a 208T > C mutation causing an amino acid substitution at S70P in C57BL/6J mice. To investigate the differences in Cdh23 mutation-related HL in different genetic backgrounds, we used the CRISPR/Cas9 system to generate homozygous mice in the CBA/CaJ background that have the same base pair missense mutation (208T > C) (Cdh23erl2/erl2 ) as Cdh23erl/erl mice in the C57BL/6J background or a single base pair deletion (235G) (Cdh23V2J2/V2J2 ) in the Cdh23 gene at exon 5. The two mutant mice exhibit hearing impairment across a broad range of frequencies. The progression of HL in Cdh23erl2/erl2 mice is slower than that in Cdh23erl/erl mice. We also found structural abnormalities in the stereocilia of cochlear hair cells in Cdh23erl2/erl2 and Cdh23V2J2/V2J2 mice. Cdh23V2J2/V2J2 mice show signs of vestibular dysfunction in open field behavior and swimming tests. In addition, we observed hair bundle defects in vestibular hair cells in Cdh23V2J2/V2J2 mice. Our results suggest an interaction between the erl locus and the C57BL/6J background that exacerbates HL in Cdh23erl/erl mice. Moreover, our study confirms that the Cdh23 gene is essential for normal hearing and balance. These two novel mutant mouse strains provide excellent models for studying CDH23 mutation-related deafness in humans.

Autophagy impairment as a key feature for acetaminophen-induced ototoxicity.

Macroautophagy/autophagy is a highly conserved self-digestion pathway that plays an important role in cytoprotection under stress conditions. Autophagy is involved in hepatotoxicity induced by acetaminophen (APAP) in experimental animals and in humans. APAP also causes ototoxicity. However, the role of autophagy in APAP-induced auditory hair cell damage is unclear. In the present study, we investigated autophagy mechanisms during APAP-induced cell death in a mouse auditory cell line (HEI-OC1) and mouse cochlear explant culture. We found that the expression of LC3-II protein and autophagic structures was increased in APAP-treated HEI-OC1 cells; however, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence, and the activity of lysosomal enzymes decreased in APAP-treated HEI-OC1 cells. The degradation of p62 protein and the expression of lysosomal enzymes also decreased in APAP-treated mouse cochlear explants. These data indicate that APAP treatment compromises autophagic degradation and causes lysosomal dysfunction. We suggest that lysosomal dysfunction may be directly responsible for APAP-induced autophagy impairment. Treatment with antioxidant N-acetylcysteine (NAC) partially alleviated APAP-induced autophagy impairment and apoptotic cell death, suggesting the involvement of oxidative stress in APAP-induced autophagy impairment. Inhibition of autophagy by knocking down of Atg5 and Atg7 aggravated APAP-induced ER and oxidative stress and increased apoptotic cell death. This study provides a better understanding of the mechanism responsible for APAP ototoxicity, which is important for future exploration of treatment strategies for the prevention of hearing loss caused by ototoxic medications.

Role of Endoplasmic Reticulum Stress in Otitis Media.

Endoplasmic reticulum (ER) stress occurs in many inflammatory responses. Here, we investigated the role of ER stress and its associated apoptosis in otitis media (OM) to elucidate the mechanisms of OM and the signaling crosstalk between ER stress and other cell damage pathways, including inflammatory cytokines and apoptosis. We examined the expression of inflammatory cytokine- and ER stress-related genes by qRT-PCR, Western blotting, and immunohistochemistry (IHC) in the middle ear of C57BL/6J mice after challenge with peptidoglycan polysaccharide (PGPS), an agent inducing OM. We also evaluated the effect of the suppression of ER stress with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. The study revealed the upregulation of ER stress- and apoptosis-related gene expression after the PGPS treatment, specifically ATF6, CHOP, BIP, caspase-12, and caspase-3. TUDCA treatment of PGPS-treated mice decreased OM; reduced the expression of CHOP, BIP, and caspase 3; and significantly decreased the proinflammatory gene expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). These results suggest that PGPS triggers ER stress and downstream proinflammatory gene expression in OM and that inhibition of ER stress alleviates OM. We propose that ER stress plays a critical role in inflammation and cell death, leading to the development of OM and points to ER stress inhibition as a potential therapeutic approach for the prevention of OM.

New insights on repeated acoustic injury: Augmentation of cochlear susceptibility and inflammatory reaction resultant of prior acoustic injury.

In industrial and military settings, individuals who suffer from one episode of acoustic trauma are likely to sustain another episode of acoustic stress, creating an opportunity for a potential interaction between the two stress conditions. We previously demonstrated that acoustic overstimulation perturbs the cochlear immune environment. However, how the cochlear immune system responds to repeated acoustic overstimulation is unknown. Here, we used a mouse model to investigate the cochlear immune response to repeated stress. We reveal that exposure to an intense noise at 120 dB SPL for 1 h activates the cochlear immune response in a time-dependent fashion with substantial expansion and activation of the macrophage population in the cochlea at 2-days post-exposure. At 20-days post-exposure, the number and pro-inflammatory phenotypes of cochlear macrophages have significantly subsided, but have yet to return to homeostatic levels. Monocytes with anti-inflammatory phenotypes are recruited into the cochlea. With the presence of this residual immune activation, a second exposure to the same noise provokes an exaggerated inflammatory response as evidenced by exacerbated maturation of macrophages. Furthermore, the second noise causes greater sensory cell pathogenesis. Unlike the first noise-induced damage that occurs mainly between 0 and 2 days post-exposure, the second noise-induced damage occurs more frequently between 2 and 20 days post-exposure, the period when secondary damage takes place. These observations suggest that repeated acoustic overstimulation exacerbates cochlear inflammation and secondary sensory cell pathogenesis. Together, our results suggest that the cochlear immune system plays an important role in modulating cochlear responses to repeated acoustic stress.

An Age-Related Hearing Protection Locus on Chromosome 16 of BXD Strain Mice.

Inbred mouse models are widely used to study age-related hearing loss (AHL). Many genes associated with AHL have been mapped in a variety of strains. However, little is known about gene variants that have the converse function-protective genes that confer strong resistance to hearing loss. Previously, we reported that C57BL/6J (B6) and DBA/2J (D2) strains share a common hearing loss allele in Cdh23. The cadherin 23 (Cdh23) gene is a key contributor to early-onset hearing loss in humans. In this study, we tested hearing across a large family of 54 BXD strains generated from B6 to D2 crosses. Five of 54 strains maintain the normal threshold (20 dB SPL) even at 2 years old-an age at which both parental strains are essentially deaf. Further analyses revealed an age-related hearing protection (ahp) locus on chromosome 16 (Chr 16) at 57~76 Mb with a maximum LOD of 5.7. A small number of BXD strains at 2 years with good hearing correspond roughly to the percentage of humans who have good hearing at 90 years old. Further studies to define candidate genes in the ahp locus and related molecular mechanisms involved in age-related resilience or resistance to AHL are warranted.

Lower level noise exposure that produces only TTS modulates the immune homeostasis of cochlear macrophages.

Noise exposure producing temporary threshold shifts (TTS) has been demonstrated to cause permanent changes to cochlear physiology and hearing function. Several explanations have been purported to underlie these long-term changes in cochlear function, such as damage to sensory cell stereocilia and synaptic connections between sensory cells and their innervation by spiral ganglion neurons, and demyelination of the auditory nerve. Though these structural defects have been implicated in hearing difficulty, cochlear responses to this stress damage remains poorly understood. Here, we report the activation of the cochlear immune system following exposure to lower level noise (LLN) that causes only TTS. Using multiple morphological, molecular and functional parameters, we assessed the responses of macrophages, the primary immune cell population in the cochlea, to the LLN exposure. This study reveals that a LLN that causes only TTS increases the macrophage population in cochlear regions immediately adjacent to sensory cells and their innervations. Many of these cells acquire an activated morphology and express the immune molecules CCL2 and ICAM1 that are important for macrophage inflammatory activity and adhesion. However, LLN exposure reduces macrophage phagocytic ability. While the activated morphology of cochlear macrophages reverses, the complete recovery is not achieved 2 months after the LLN exposure. Taken together, these observations clearly implicate the cochlear immune system in the cochlear response to LLN that causes no permanent threshold change.

Differential fates of tissue macrophages in the cochlea during postnatal development.

The cochlea contains macrophages. These cells participate in inflammatory responses to cochlear pathogenesis. However, it is not clear how and when these cells populate the cochlea during postnatal development. The current study aims to determine the postnatal development of cochlear macrophages with the focus on macrophage development in the organ of Corti and the basilar membrane. Cochleae were collected from C57BL/6J mice at ages of postnatal day (P) 1 to P21, as well as from mature mice (1-4 months). Macrophages were identified based on their expression of F4/80 and Iba1, as well as their unique morphologies. Two sets of macrophages were identified in the regions of the organ of Corti and the basilar membrane. One set resides on the scala tympani side of the basilar membrane. These cells have a round shape at P1 and start to undergo site-specific differentiation at P4. Apical macrophages adopt a dendritic shape. Middle and basal macrophages take on an irregular shape with short projections. Basal macrophages further differentiate into an amoeboid shape. The other set of macrophages resides above the basilar membrane, either beneath the cells of the organ of Corti or along the spiral vessel of the basilar membrane. As the sensory epithelium matures, these cells undergo developmental death with the phenotypes of apoptosis. Macrophages are also identified in the spiral ligament, spiral limbus, and neural regions. Their numbers decrease during postnatal development. Together, these results suggest a dynamic rearrangement of the macrophage population during postnatal cochlear development.

Immune cells and non-immune cells with immune function in mammalian cochleae.

The cochlea has an immune environment dominated by macrophages under resting conditions. When stressed, circulating monocytes enter the cochlea. These immune mediators, along with cochlear resident cells, organize a complex defense response against pathological challenges. Since the cochlea has minimal exposure to pathogens, most inflammatory conditions in the cochlea are sterile. Although the immune response is initiated for the protection of the cochlea, off-target effects can cause collateral damage to cochlear cells. A better understanding of cochlear immune capacity and regulation would therefore lead to development of new therapeutic treatments. Over the past decade, there have been many advances in our understanding of cochlear immune capacity. In this review, we provide an update and overview of the cellular components of cochlear immune capacity with a focus on macrophages in mammalian cochleae. We describe the composition and distribution of immune cells in the cochlea and suggest that phenotypic and functional characteristics of macrophages have site-specific diversity. We also highlight the response of immune cells to acute and chronic stresses and comment on the potential function of immune cells in cochlear homeostasis and disease development. Finally, we briefly review potential roles for cochlear resident cells in immune activities of the cochlea.

Loss of sestrin 2 potentiates the early onset of age-related sensory cell degeneration in the cochlea.

Sestrin 2 (SESN2) is a stress-inducible protein that protects tissues from oxidative stress and delays the aging process. However, its role in maintaining the functional and structural integrity of the cochlea is largely unknown. Here, we report the expression of SESN2 protein in the sensory epithelium, particularly in hair cells. Using C57BL/6J mice, a mouse model of age-related cochlear degeneration, we observed a significant age-related reduction in SESN2 expression in cochlear tissues that was associated with early onset hearing loss and accelerated age-related sensory cell degeneration that progressed from the base toward the apex of the cochlea. Hair cell death occurred by caspase-8 mediated apoptosis. Compared to C57BL/6J control mice, Sesn2 KO mice displayed enhanced expression of proinflammatory genes and activation of basilar membrane macrophages, suggesting that loss of SESN2 function provokes the immune response. Together, these results suggest that Sesn2 plays an important role in cochlear homeostasis and immune responses to stress.

Prolonged low-level noise-induced plasticity in the peripheral and central auditory system of rats.

Prolonged low-level noise exposure alters loudness perception in humans, presumably by decreasing the gain of the central auditory system. Here we test the central gain hypothesis by measuring the acute and chronic physiologic changes at the level of the cochlea and inferior colliculus (IC) after a 75-dB SPL, 10-20-kHz noise exposure for 5weeks. The compound action potential (CAP) and summating potential (SP) were used to assess the functional status of the cochlea and 16 channel electrodes were used to measure the local field potentials (LFP) and multi-unit spike discharge rates (SDR) from the IC immediately after and one-week post-exposure. Measurements obtained immediately post-exposure demonstrated a significant reduction in supra-threshold CAP amplitudes. In contrast to the periphery, sound-evoked activity in the IC was enhanced in a frequency-dependent manner consistent with models of enhanced central gain. Surprisingly, one-week post-exposure supra-threshold responses from the cochlea had not only recovered, but were significantly larger than normal, and thresholds were significantly better than controls. Moreover, sound-evoked hyperactivity in the IC was sustained within the noise exposure frequency band but suppressed at higher frequencies. When response amplitudes representing the neural output of the cochlea and IC activity at one-week post exposure were compared with control animal responses, a central attenuation phenomenon becomes evident, which may play a key role in understanding why low-level noise can sometimes ameliorate tinnitus and hyperacusis percepts.

Involvement of p53 and Bcl-2 in sensory cell degeneration in aging rat cochleae.

CONCLUSION: p53 and Bcl-2 (B-cell lymphoma 2) are involved in the process of sensory cell degeneration in aging cochleae. OBJECTIVE: To determine molecular players in age-related hair cell degeneration, this study examined the changes in p53 and Bcl-2 expression at different stages of apoptotic and necrotic death of hair cells in aging rat cochleae. METHODS: Young (3-4 months) and aging (23-24 months) Fisher 344/NHsd rats were used. The thresholds of the auditory brainstem response (ABR) were measured to determine the auditory function. Immunolabeling was performed to determine the expression of p53 and Bcl-2 proteins in the sensory epithelium. Propidium iodide staining was performed to determine the morphologic changes in hair cell nuclei. RESULTS: Aging rats exhibited a significant elevation in ABR thresholds at all tested frequencies (p < 0.001). The p53 and Bcl-2 immunoreactivity was increased in aging hair cells showing the early signs of apoptotic changes in their nuclei. The Bcl-2 expression increase was also observed in hair cells displaying early signs of necrosis. As the hair cell degenerative process advanced, p53 and Bcl-2 immunoreactivity became reduced or absent. In the areas where no detectable nuclear staining was present, p53 and Bcl-2 immunoreactivity was absent.

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status.