Lack of Fractalkine Receptor on Macrophages Impairs Spontaneous Recovery of Ribbon Synapses After Moderate Noise Trauma in C57BL/6 Mice

Abstract

Noise trauma causes loss of synaptic connections between cochlear inner hair cells (IHCs) and the spiral ganglion neurons (SGNs). Such synaptic loss can trigger slow and progressive degeneration of SGNs. Macrophage fractalkine signaling is critical for neuron survival in the injured cochlea, but its role in cochlear synaptopathy is unknown. Fractalkine, a chemokine, is constitutively expressed by SGNs and signals via its receptor CX₃CR1 that is expressed on macrophages. The present study characterized the immune response and examined the function of fractalkine signaling in degeneration and repair of cochlear synapses following noise trauma. Adult mice wild type, heterozygous and knockout for CX₃CR1 on a C57BL/6 background were exposed for 2 h to an octave band noise at 90 dB SPL. Noise exposure caused temporary shifts in hearing thresholds without any evident loss of hair cells in CX₃CR1 heterozygous mice that have intact fractalkine signaling. Enhanced macrophage migration toward the IHC-synaptic region was observed immediately after exposure in all genotypes. Synaptic immunolabeling revealed a rapid loss of ribbon synapses throughout the basal turn of the cochlea of all genotypes. The damaged synapses spontaneously recovered in mice with intact CX₃CR1. However, CX₃CR1 knockout (KO) animals displayed enhanced synaptic degeneration that correlated with attenuated suprathreshold neural responses at higher frequencies. Exposed CX₃CR1 KO mice also exhibited increased loss of IHCs and SGN cell bodies compared to exposed heterozygous mice. These results indicate that macrophages can promote repair of damaged synapses after moderate noise trauma and that repair requires fractalkine signaling.

Keywords: C57BL/6 mice; cochlea; fractalkine; macrophages; noise-induced hearing loss; ribbon synapses.

FIGURE 1

Temporary noise-induced hearing loss causes synaptopathy. (A) Auditory brainstem response thresholds plotted as a function of stimulus frequencies obtained from CX₃CR1^+/- mice prior to noise exposure (baseline), and at 0 h and 2 weeks after noise exposure. n = 9, ^∗p < 0.05 baseline vs. 0 h, ^∗∗∗∗p < 0.0001 baseline vs. 0 h and 2 weeks. (B) DPgrams obtained from CX₃CR1^+/- mice prior to noise exposure (baseline), and at 0 h and 2 weeks after noise exposure. n = 6, ^∗∗∗∗p < 0.0001 baseline vs. 0 h, ^∗∗p = 0.0046 baseline vs. 2 weeks (at 40 kHz). Open black circles represents noise floor. (C) Representative micrographs of cochlear whole mounts from apical, middle and basal regions of CX₃CR1^+/- mice not exposed to noise (no exposure) and at time 0 h, 2 and 8 weeks recovery after exposure immunolabeled for hair cell marker, Myosin VIIa (red). (D) Representative micrograph from unexposed CX₃CR1^+/- mouse cochlea at 32 kHz region immunolabeled for pre-synaptic marker CtBP2 (red), post-synaptic marker GluA3 (green) and inner hair cell marker Myosin VIIa (blue) showing intact juxtaposed ribbon synapses. (E) Representative micrograph from CX₃CR1^+/- mouse cochlea at 24 h after exposure from 32 kHz region showing disintegrated ribbon synapses indicated by white arrow. (F) Representative micrograph from CX₃CR1^+/- mouse cochlea at 2 weeks after exposure from 32 kHz region showing synaptic repair. (G) Representative micrograph from CX₃CR1^+/- mouse cochlea at 8 weeks after exposure from 32 kHz region showing stable synapses. Labeling colors in (E–G) are same as reported in (D). (H) Ribbon synapses per IHC along the cochlear length, n = 6–9 mice per time point, ^∗∗∗∗p < 0.0001, 24 h vs. control. Gray bar in the graph represents the frequency band (8–16 kHz) of noise exposure. Scale bar, 63 μm (C) and 17 μm (D–G).

FIGURE 2

Macrophage migrate into the epithelium after noise trauma. Representative cochlear whole mount micrographs from CX₃CR1^+/- mice (A) not exposed to noise (no exposure), (B) at 0 h, and (C) 2 weeks after noise exposure immunolabeled for macrophages (green, GFP) and peripheral nerve fibers (red, NF165 and Tuj-1). (D) Macrophage numbers per 100 μm of sensory epithelium. No exposure (n = 5), 0 h (n = 11) and 2 weeks (n = 7). ^∗p < 0.05, ^∗∗p < 0.01, control vs. 2 weeks. (E) The organ of Corti from an unexposed mouse (no exposure) showing macrophages in the osseous spiral lamina away from the IHC-synaptic region. (F) GFP-macrophage observed in the basal region of an inner hair cell (Myosin VIIa, blue) at 0 h after noise exposure. (G) Higher magnification of (F) showing GFP-macrophage above the habenula and in the basal region of the IHC at 0 h after synaptopathic noise exposure. White color in E-G represents nuclei stained with DAPI. (H) Macrophage numbers in the IHC basal region per 100 μm of epithelium. No exposure (n = 5), 0 h (n = 11) and 2 weeks (n = 7).^∗∗∗p < 0.001 (16–28 kHz), no exposure vs. 0 h, ^∗∗∗∗p < 0.0001 (5–11, 16–28 kHz), ^∗p < 0.05 (32–45 kHz), no exposure vs. 2 weeks. (I,J) 3D reconstruction of a confocal stack showing pseudopodia (cytoplasmic extension) of a GFP-macrophage above the habenula and under the basal pole (BP) of an inner hair cell (IHC, blue) and the cell body of the macrophage in the osseous spiral lamina (OSL). Dashed line represents demarcation of the organ of Corti from the OSL. Scale bar, 63 μm (A–C), 20 μm (E,F), 10 μm (G), and 6.8 μm (I,J).

FIGURE 3

Auditory brainstem responses. (A–C) Audiograms of CX₃CR1^+/+ (black), CX₃CR1^+/- (blue) and CX₃CR1^-/- (red) mice not exposed to noise at 6 weeks (A), 8 weeks (B), and 14 weeks (C) of age. n = 5–6 mice per genotype. (D–F) Audiograms of CX₃CR1^+/+ (black), CX₃CR1^+/- (blue), and CX₃CR1^-/- (red) mice exposed to noise at time 0 h (D), 2 weeks (E), and 8 weeks (F) recovery. Filled circles with solid lines represent thresholds prior to noise-exposure (Pre-NE) and unfilled circles with hyphenated lines represents thresholds post-noise exposure (Post-NE). CX₃CR1^+/+ (n = 7), CX₃CR1^+/- (n = 9), CX₃CR1^-/- (n = 15). Gray bar in the graph represents the frequency band (8–16 kHz) of noise exposure.

FIGURE 4

Distortion product otoacoustic emissions. DP grams of CX₃CR1^+/+ (black), CX₃CR1^+/- (blue), and CX₃CR1^-/- (red) mice exposed to noise at time 0 h (A), 2 weeks (B), and 8 weeks (C) recovery. Filled circles with solid lines represent DP grams prior to noise-exposure (Pre-NE) and unfilled circles with hyphenated lines represents DP grams post-noise exposure (Post-NE). CX₃CR1^+/+ (n = 8), CX₃CR1^+/- (n = 9), CX₃CR1^-/- (n = 9). Gray bar in the graph represents the frequency band (8–16 kHz) of noise exposure.

FIGURE 5

ABR Wave 1 amplitudes against level series. (A) Representative ABR trace (28.3kHz, 80 dB SPL) showing placement of arrows at peak and trough of ABR wave 1 for amplitude measurements. ABR wave 1 amplitudes at 28.3 kHz from CX₃CR1^+/+ (black), CX₃CR1^+/- (blue), and CX₃CR1^-/- (red) mice exposed to noise at time 0 h (B–B″), 2 weeks (C–C″), and 8 weeks (D–D″) recovery. Filled circles with solid lines represent amplitudes prior to noise-exposure (Pre-NE) and unfilled circles with hyphenated lines represents amplitudes post-noise exposure (Post-NE). CX₃CR1^+/+ (n = 7), CX₃CR1^+/- (n = 9), CX₃CR1^-/- (n = 15).

FIGURE 6

Ribbon synapses, macrophages, and hair cell numbers in CX₃CR1^+/- and CX₃CR1^-/- mice after noise exposure. (A) Representative micrograph from CX₃CR1^+/- mice not exposed to noise (no exposure) at 32 kHz region immunolabeled for pre-synaptic marker CtBP2 (red), post-synaptic marker GluA3 (green) and inner hair cell marker Myosin VIIa (blue) showing intact juxtaposed ribbon synapses. (B) Representative micrograph at 24 h after exposure from CX₃CR1^+/- mice at 32 kHz region showing disintegrated ribbon synapses. (C) Representative micrograph at 8 weeks after exposure from CX₃CR1^+/- mice at 32 kHz region showing stable repaired synapses. (D) Representative micrograph at 8 weeks after exposure from CX₃CR1^-/- mice at 32 kHz region showing degenerated ribbon synapses. Labeling colors in panels (B–D) are same as reported in A. (E) Ribbon synapses per surviving IHC along the cochlear frequency regions. No exposure; n = 6–7 mice per genotype, 24 h after exposure; n = 5–6 mice per genotype, 8 weeks after exposure; n = 9–15 mice per genotype. ^∗∗p < 0.01 (CX₃CR1^+/- and CX₃CR1^-/-, no exposure vs. 24 h), #p < 0.01 (CX₃CR1^-/-, 8 weeks vs. no exposure). Gray bar in the graphs represents the frequency band (8–16 kHz) of noise exposure. (F) Average macrophage numbers in IHC synaptic region per 100 μm of sensory epithelium of exposed CX₃CR1^+/- and CX₃CR1^-/- mice, n = 9 per genotype, ns, not significant. Percentage outer hair cell survival (G) and inner hair cell survival (H). Mice per genotype not exposed to noise (n = 6), CX₃CR1^+/- (n = 9), CX₃CR1^-/- (n = 15) at 8 weeks after exposure. ^∗∗p < 0.01. Scale, 17 μm (A-D).

FIGURE 7

Macrophage and Spiral ganglion neuron density in the spiral ganglion of CX₃CR1^+/- and CX₃CR1^-/- mice after exposure. Representative micrographs of cochlear mid-modiolar sections from CX₃CR1^+/- mice not exposed to noise (no exposure) (A) and from CX₃CR1^+/- mice at 24 h (B), 2 weeks (C), and 8 weeks (D) after exposure immunolabeled for macrophages (green, GFP) and spiral ganglion neurons (red, NF165 and Tuj-1). (E) Macrophage numbers in spiral ganglion (SG) (per 1,000 μm²), n = 4–8 CX₃CR1^+/- mice per recovery time point. Representative micrographs showing fewer SGN cell bodies immunolabeled for NF165 and Tuj-1 (red) in CX₃CR1^-/- mice (I) compared to CX₃CR1^+/- mice (H) at 24 weeks post-exposure and to unexposed age-matched control mice (F,G). (J) Quantitative data on SGN density per 1,000 μm² from age-matched unexposed and exposed CX₃CR1^+/- and CX₃CR1^-/- mice at 24 weeks post-noise trauma. n = 4 mice per group, ^∗∗p < 0.01 CX₃CR1^-/- mice 24 weeks post-NE vs. no exposure CX₃CR1^+/- and CX₃CR1^-/- mice. (K) Average macrophage density in SG of CX₃CR1^+/- and CX₃CR1^-/- mice at time 24 h, 2, 8, 16, and 24 weeks recovery after exposure. n = 4–9 per genotype per recovery time point. Scale bar, 63 μm.