Age-Related Hearing Loss Is Accompanied by Chronic Inflammation in the Cochlea and the Cochlear Nucleus

Abstract

Age-related hearing loss (ARHL) is a major hearing impairment characterized by pathological changes in both the peripheral and central auditory systems. Low-grade inflammation was observed in the cochlea of deceased human subjects with ARHL and animal models of early onset ARHL, which suggests that inflammation contributes to the development of ARHL. However, it remains elusive how chronic inflammation progresses during normal aging in the cochlea, and especially the accompanying changes of neuroinflammation in the central auditory system. To address this, we investigated chronic inflammation in both the cochlea and the cochlear nucleus (CN) of CBA/CaJ mice, an inbred mouse strain that undergoes normal aging and develops human, like-late-onset ARHL. Using immunohistochemistry, confocal microscopy, and quantitative image processing, we measured the accumulation and activation of macrophages in the cochlea and microglia in the CN using their shared markers: ionized calcium binding adaptor molecule 1 (Iba1) and CD68-a marker of phagocytic activity. We found progressive increases in the area covered by Iba1-labeled macrophages and enhanced CD68 staining in the osseous spiral lamina of the cochlea that correlated with elevated ABR threshold across the lifespan. During the process, we further identified significant increases in microglial activation and C1q deposition in the CN, indicating increased neuroinflammation and complement activation in the central auditory system. Our study suggests that during normal aging, chronic inflammation occurs in both the peripheral and the central auditory system, which may contribute in coordination to the development of ARHL.

Keywords: aging; cochlea; cochlear nucleus; complement; hearing loss; inflammation; macrophage; microglia.

Figure 1

ARHL-associated inflammation increases across the lifespan and correlates with the severity of hearing impairment in CBA/CaJ mice. (A–C) Representative images of young, middle-aged, and aged mice showing accumulation of cochlear macrophages in the OSL, scale bar = 200 μm, inserts scale bar = 10 μm. Myosin VIIA staining revealed minimal OHC loss (arrows) in middle-aged mice, while few OHC remained (triangles) in aged mice. (D) Significant differences were found between all age groups. ***p < 0.001, ****p < 0.0001. (E) ABR Threshold increases with age confirming late-onset ARHL. ***p < 0.001. (F) Regression analysis between macrophage area (μm²/100 μm²) and ABR Threshold to clicks (dB) reveals a significant linear correlation R² = 0.9081, p < 0.002.

Figure 2

CD68 levels increase in OSL across aging. (A–C) Representative images of CD68 labeling in the OSL (ROI indicated by white dashed line) of young, middle-aged, and aged mice. Note: occasionally the tectorial membrane was not completely removed adding noise in the signal. This noise was excluded from ROI analysis (see ROI in panel C). Scale bar = 200 μm. Arrows: indicate Iba1+ CD68⁺ macrophages. Triangles: indicate CD68 outside Iba1⁺ macrophages. (D) Quantification of CD68 shows significant increases across aging. *p < 0.05. (E) Co-localization of CD68 and Iba1 shows a trend toward increases in intracellular CD68 inside Iba1-labeled macrophages across aging. ^#p < 0.1. (F) Quantification of CD68 outside Iba1-labeled macrophages shows an increase in Iba1⁻ CD68⁺ only in aged mice possibly indicating increased infiltration. *p < 0.05. ns: not significant.

Figure 3

Age-associated microglia activation in the CN. (A) Representative images showing the staining of Iba1, CD68, P2Y12, and the overlapped image of Iba1 and CD68 in the AVCN of young mice. Dashed line marks the AVCN area for analysis; scale bar: 200 μm. (B) Magnified view of the ROI from (A); scale bar: 25 μm. (C–F) Same images obtained from middle-aged mice (C,D) and aged mice (E,F). Triangles mark: microglia with activation-associated morphology; arrows: microglia with major downregulation P2Y12; and stars: microglia with increased CD68 content. Importantly, these three approaches to identify activation capture various populations, suggesting heterogeneity of microglial activation in aged mice. Notice, for example, one of two microglia with downregulated P2Y12 (top arrow) also had increased CD68 staining, while the other does not. (G) Schematic of the CN. Dashed square mark AVCN as shown in (A,C,E). (H) Summary changes of Iba1- and CD68-labeled area during aging. Iba1-labeled area did not significantly differ between groups, while CD68 increased with age. One-way ANOVA: ***p < 0.001. ns: not significant. (I) P2Y12 is downregulated during late-stage ARHL when the most dramatic increase in phagocytic activation occurs as quantified by colocalized area μm²/100 μm² CD68/Iba1. One-way ANOVA: **p < 0.01; ****p < 0.0001.

Figure 4

C1q deposition in the CN dramatically increases during ARHL. (A–C) Representative images to show that C1q-IR is progressively increased throughout the CN during aging. Scale bar = 200 μm. (D–F) High resolution confocal imaging reveals intracellular C1q inside microglia dramatically increases in aging. Arrows: C1q deposited on tissue outside of microglia; Stars: internal C1q inside microglia. Scale bar = 25 μm. (G) Quantification of C1q-IR by mean fluorescence intensity analyzed by ImageJ shows significantly increased C1q-IR across the lifespan. **p < 0.01; ****p < 0.0001. (H) C1q levels rise inside microglia across aging. *p < 0.05. (I) The total amount of C1q deposition increases with age. *p < 0.05; ***p < 0.001.