Neural presbycusis at ultra-high frequency in aged common marmosets and rhesus monkeys

ABR thresholds at ultra-high frequencies in geriatric marmosets

ABRs to tones were recorded in 38 marmosets aged 2 years and 1 month to 10 years and 5 months. Marmosets age at a rate approximately seven times faster than humans, so these animals were roughly equivalent humans aged 14 to 70 years. Because auditory sensitivity in these animals became weaker at frequencies >32 kHz, hearing at up to 32 kHz was tested in these animals [28].

The ABR waveform during the first 10ms after a stimulus consisted of five positive waves (I-V), with five to six positive waves detected at high sound pressure (Figure 1A–1D). Not all of these components were detected in all animals. ABR morphology did not differ significantly between male and female marmosets. Comparisons of the size and latency of the peaks in each trace from top to bottom showed that, as the stimulus level decreased, the amplitude of the peaks decreased and the latency increased. ABR wave amplitude was lower at 32 kHz than at 8 kHz in both young (Figure 1A, 1B) and geriatric (Figure 1C, 1D) marmosets. Aging did not affect ABR at 8 kHz, but the ABR waveform was not reliable at 32 kHz in the 10-year-old marmoset (Figure 1B, 1D).

Figure 1. Effects of age on ABR thresholds in marmosets. (A–D) Examples of ABR waveforms of common marmosets aged 2 and 10 years following 80 dB SPL tone bursts at frequencies of 8 and 32 kHz. (E, F) Scatter plots and corresponding regression lines and regression equations for the relationships between ABR thresholds (dB SPL) of 36 individual marmosets and age (years) at frequencies of tone burst stimuli of 8 kHz (E) R-squared linear=0.01168, P=0.5304) and 32 kHz (F) R-squared linear=0.3172, P=0.0003). (G) Relationships between ages and auditory thresholds determined by ABR. Data are presented as mean ± SEM. **P < 0.01; ****P < 0.0001 by two-way ANOVA for significant differences in ABR thresholds between 2-4-year-old and 8-10-year-old marmosets; *P < 0.05, ***P < 0.001 by two-way ANOVA for significant differences in ABR thresholds between 5-7-year-old and 8-10-year-old animals.

ABR component II was elicited firstly and distinctly in common marmoset [28], so the ABR threshold was defined as the lowest intensity of each specified stimulus that generated an observable wave II. Comparisons of the ABR thresholds of all monkeys at stimuli of 2, 4, 8, 16, 22 and 32 kHz showed that ABR thresholds were not correlated with age at low frequencies (Figure 1E and Supplementary Figure 1), but there were positively correlations with age at 16 kHz (F=4.326, R²=0.1129, p=0.0451) and 32 kHz (F=15.80, R²=0.3172, p=0.0003; Figure 1F). A regression model revealed a rate of increase of 4.1 dB per year for ABR thresholds at 32 kHz, indicating that ABR thresholds increased with age at ultra-high frequencies in common marmosets, unlike age-related ABR thresholds increasing from 0.5-16 kHz in rhesus monkeys [37].

To analyze the associations of ABR thresholds with age individually, the 38 marmosets were divided into three groups, young (2-4 years of age, 20 animals), middle-aged (5-7 years of age, 11 animals), and geriatric (8-10 years of age, 7 animals) marmosets. Significant differences among these three groups were not observed at thresholds of 2-8 kHz, except that the young and geriatric groups differed at 2 kHz (Figure 1E). Age-related hearing loss was first observed at 16 kHz, at which the ABR threshold was 18 dB (p<0.01) higher in geriatric than in middle-aged marmosets, indicating that hearing sensitivity decreased with age. At 22 kHz, geriatric marmosets exhibited ABR thresholds 17 dB higher than in young animals (p<0.005) and 21 dB higher than in middle-aged animals (p<0.001). ABR thresholds at 32 kHz were 26 dB higher in geriatric than in young animals and 16 dB higher in geriatric than in middle-aged animals.

Decreased age-related ABR amplitude at ultra-high frequency

To compare ABR peaks at a suprathreshold sound level in the three groups of monkeys, wave II was analyzed at 80 dB SPL across all of the stimuli from 2 to 32 kHz. The value of 80 dB SPL was chosen because ABR waveforms were distinct in most of monkeys studied, and peak II was chosen to analyze age-related changes because geriatric monkeys required higher stimulus levels than the other age groups to elicit peaks II and IV [37].

Age was calculated to one decimal place in years, and the correlation between age and ABR amplitude was analyzed. Regression equations showed that the ABR amplitude did not change with age at 2-22 kHz, with R² values ranging between 0.0023 and 0.0722 (P>0.05 each; Figure 2A–2E). Age-related reductions in ABR amplitude were observed at 32 kHz (F=4.638, R²=0.1200, p=0.0385), with the amplitudes decreasing 0.07954 μV per year (Figure 2F). Peak amplitudes were lower at ultra-high frequency (32 kHz) than at low and middle frequencies (Figure 2).

Figure 2. Effects of age on ABR amplitude at ultra-high frequency in marmosets. (A–F) Scatter plots and corresponding regression lines and regression equations for the relationships between ABR amplitudes (μV) of 36 individual marmosets and age (years) at frequencies of 2 kHz (A; R-squared linear=0.0023, P=0.7789); 4 kHz (B; R-squared linear=0.0722, P=0.1129); 8 kHz (C; R-squared linear=0.0138, P=0.4944); 16 kHz (D; R-squared linear=0.05486, P=0.1691); 22 kHz (E; R-squared linear=0.1138, P=0.0442); and 32 kHz (F; R-squared linear=0.1200, P=0.0385).

The latencies of ABR component II were analyzed at all frequencies following stimulation at 80 dB SPL. However, there was no age-related difference in latency among these groups of marmosets (Supplementary Figure 2).

DPOAE threshold and amplitude did not correlate with age

DPOAEs are responses generated when the cochlea is stimulated simultaneously by two pure tone frequencies, which are similar in rhesus monkeys and humans [38–40]. To our knowledge, only two previous studies assessed DPOAEs in marmosets, finding that DPOAEs tended to be slightly stronger in the right than in the left ear [41, 42]. Neither of these studies, however, analyzed the associations between DPOAE and age in marmosets.

DPOAEs were detected in 44 ears of marmosets at different frequencies (2-32 kHz), with 6 animals showing the occurrence of DPOAEs in both ears. The threshold was defined as the last detectable amplitude following auditory stimulation. Averaged DPOAEs did not vary distinctly at all frequencies, ranging from 53.3-66.1 dB SPL Figure 3A), although DPOAE thresholds were determined to be 40 dB SPL in several individual animals. Unlike ABR, DPOAE thresholds at all frequencies tested did not correlate with age (Supplementary Figure 3, p>0.05 each).

Figure 3. DPOAE threshold and amplitude were not affected by age. (A) Association between age and auditory thresholds determined by DPOAE. Data are presented as mean ± SEM. No significant differences were observed. (B–G) Scatter plots and corresponding regression lines and regression equations for the relationship between the DPOAE amplitudes (dB SPL) of 51 ears in 36 individual marmosets and age (years) at tone burst stimuli of 2 kHz (B; R-squared linear=0.02408, P=0.2769); 4 kHz (C; R-squared linear=0.00085, P=0.8391); 8 kHz (D; R-squared linear=0.02160, P=0.3035); 16 kHz (E; R-squared linear=0.07479, P=0.0522); 22 kHz (F; R-squared linear=0.00754, P=0.5446); and 32 kHz (G; R-squared linear=0.00488, P=0.6263).

When the tested animals were divided into three age groups, as described in the ABR test, there were no significant difference in DPOAE thresholds among these three groups (Figure 3A), indicating that OHC function did not worsen with age in marmosets. Analysis of DPOAE amplitudes at different frequencies also did not differ by age (Figure 3B–3G, p>0.05 each).

Auditory periphery neural degeneration in geriatric common marmosets

To explore the mechanism of age-related ABR changes in common marmosets, the cochleae were extracted from the temporal bone of a 2-year-old marmoset. Whole mount preparations of the cochlea revealed hair cells in the organ of Corti. Hair cells (HCs), supporting cells and neural connections in the cochlea were labeled immunohistologically with antibodies to MYO7A, SOX2 and NF-H, respectively (Figure 4A–4A’’’). This methodology, however, could not show all putative myelinated spiral ganglion cell bodies. To assess SGN associated neural presbycusis, the morphological features of basal-modiolus semi-thin sections from a 2- year-old and a 12-year-old marmoset were compared.

Figure 4. Effects of age on SGN density in marmosets. (A–A”) Confocal whole-mount immunofluorescence images of the middle turn of a cochlea in a 2-year-old marmoset. (A) Hair cells labeled with MYO7A. (A’) Supporting cells positive for SOX2. (A”) Neural fibers labeled with NF-H. (A”’) Merged channels figure, MYO7A (green), SOX2 (red), NF-H (magenta), DAPI (blue). (B–B”) Confocal images of a cochlear cryosection from a 2-year-old marmoset. SGNs labeled with TUJ. TUJ-positive neurofilaments connect the basilar membrane (BM) to the modiolus. (C–C”) Confocal images of a cochlear cryosection from a 12-year-old marmoset. Scale bar: 100 μm.

The basilar membrane (BM) morphology did not differ in the inner ears of these two marmosets (Figure 4B, 4C). TUJ1 antibody, which reacts with beta-tubulin III, was found to stain SGN in developing marmosets [30]. The packing density of SGNs in the modiolus was much higher in the 2-year-old than in the 12-year-old marmoset (Figure 4B, 4C). Several non-cell cavities were observed in the modiolus of the older marmoset, indicating that the degeneration of SGNs leads to age-related hearing loss, rather than HC loss. In addition to the reduction of TUJ1-positive cells in the cochlea of the geriatric marmoset, the remaining TUJ1-positive cells were both disorganized and swollen in this animal (Figure 5A, 5B).

Figure 5. Effects of age on expression patterns of neuronal and glial markers in marmosets. Confocal immunofluorescence images of cryosections of the modiolus regions of 2-year-old and 12-year-old marmosets. SGNs labeled with TUJ in the (A, A’) 2-year-old and (B, B’) 12-year-old marmosets. MAP2 positive neurons in the modiolus of the (C, C’) 2-year-old and (D, D’) 12-year-old marmosets. GFAP2 positive glia were in the (E, E’) 2-year-old and (F, F’) 12-year-old marmosets. Apoptosis was labelled by cleaved Caspase-3 in 2-year-old marmoset (G, G’) and 12-year-old marmoset (H, H’). Scale bar: 50 μm.

To better assess modiolus pathology in marmosets, thin sections from the cochlea of a geriatric marmoset were stained with antibodies to additional cell types. In a previous study in rodents, microtubule associated protein 2 (MAP2) was tightly regulated in rodent SGNs undergoing repair, suggesting that MAP2 positive cells may be involved in the regeneration of primary auditory neurons after injury [43]. Many MAP2 positive cells were observed in the cochleae of both the young and geriatric marmosets, with no distinct difference between them (Figure 5C, 5D), indicating that primates have limited SGN regeneration capacity after birth. Age-related changes in the expression of glial fibrillary acidic protein (GFAP) were observed in rodent cochlear nuclei [44], suggesting that the increase in the number of GFAP positive astrocytes may be due to alterations in auditory nerve fibers and trophic interactions with post-synaptic cells. The current study found that many GFAP positive cells were around SGNs in the young marmosets (Figure 5E), but that the number of GFAP positive cells in the modiolus was lower in the geriatric marmosets (Figure 5F). This decrease in GFAP positive cells reflected astrocyte degeneration in the cochlea, accompanying neuron degeneration.

To validate neural degeneration, we labelled cleaved caspase-3 in young and old marmoset. Caspase-3 is defined as the effector caspase that cleave key regulatory or structural proteins and in particular activate apoptotic nucleases [45] and was used to detect apoptosis SGN of rodents after ototoxicity [46, 47]. We observed plenty of cleaved caspase-3 positive cells in the modiolus of geriatric marmoset cochlea (Figure 5G), and rare cleaved caspase-3 positive cells in the young one (Figure 5H). The arising of caspase-3 positive cells in modiolus of aged marmoset indicated SGN apoptosis appeared during aging, which demonstrated neural apoptosis was the main reason for ARHL.

Auditory dys-synchrony at ultra-high frequency in geriatric rhesus monkeys

To explore auditory function at ultra-high frequencies in another NHP, DPOAE and ABR were tested in 40 rhesus monkeys aged 2 to 24 years. Rhesus monkeys age approximately three times faster than humans, making these animals roughly equivalent to humans aged 6 to 72 years. ABR results did not differ significantly in male and female rhesus monkeys (data not shown).

The 40 animals were divided into three groups: young (2-10 years of age, 13 animals), middle-aged (11-17 years of age, 16 animals), and geriatric (18-25 years of age, 18 animals). Auditory functions at 8 to 32 kHz were significantly poorer in geriatric than in young monkeys (Figure 6A). Compared with young rhesus monkeys, ABR thresholds in geriatric monkeys were 13.5, 16.5, 16.0 and 19.0 dB higher at 8 (p<0.05), 16 (p<0.01), 22 (p<0.01) and 32 (p<0.005) kHz, respectively. ABR thresholds also differed between middle-aged and geriatric monkeys, but only at ultra-high frequencies of 22 and 32 kHz (p<0.05 each), indicating that the rising ABR threshold at high frequency was incipient audiogram indicators of ARHL.

Figure 6. Effects of age on auditory function in rhesus monkeys. (A) Age-related changes in auditory thresholds determined by ABR. Data are presented as mean ± SEM. Significant differences in ABR threshold between the groups are marked. Compared to 2-10 year-old animals, ABR thresholds increased in aging rhesus monkeys from 8-32 kHz. ABR thresholds increased at 22 and 32 kHz compared aging monkeys with 11-17 y.o. ones. *P < 0.05; **P < 0.01; ***P < 0.001 by two-way ANOVA for comparisons of ABR thresholds in 2-10-year-old and 11-17-year-old rhesus monkeys. (B) Scatter plot and corresponding regression line and regression equation for the relationship between ABR amplitudes (nV) and ages (years) of 40 individual rhesus monkeys at a frequency of tone burst stimuli of 22 kHz (R-squared linear= 0.1379, P=0.0183). (C) Scatter plot and corresponding regression line and regression equation for the relationship between ABR latencies (ms) and ages (years) of the 40 rhesus monkeys at a frequency of 22 kHz (R-squared linear= 0.1010, P=0.0456). (D) Association between age and auditory thresholds determined by DPOAE in rhesus monkeys. Data are presented as mean ± SEM. *P < 0.05 by two-way ANOVA for comparisons of DPOAE thresholds in 2-10-year-old and 18-25-year-old animals.

Because ABR wave II was distinct in rhesus monkeys [37], its amplitudes and latencies were analyzed to evaluate auditory function. Linear regression analysis showed an age-related decrease in ABR amplitude of 70 dB at 22 kHz (F=6.080, R²=0.1379, p=0.0183), with a slope of -2.225 relative to age (Figure 6B). In contrast, the linear regression equation of latency under the same conditions showed a significant prolongation (F=4.271, R²=0.1010, p=0.0456), with a slope of 0.01627 (Figure 6C).

Testing of DPOAE in 15 rhesus monkeys, five from each group, showed no age-related differences in DPOAE thresholds, except at 32 kHz. The DPOAE threshold was 25.0 dB higher in geriatric than in young rhesus monkeys at 32 kHz (p<0.05), but there were no significant differences at other frequencies (Figure 6D), indicating that DPOAE threshold was stable with age in rhesus monkeys.

Subjects

Thirty-eight common marmosets (Callithrix jacchus) were obtained from the Institute of Neuroscience, the Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. The animals were aged 1 to 11 years, with an average life span in a breeding colony of around 10-12 years [57]. None of the animals in this study had behavioral disorders and all had clear ear canals and intact tympanic membranes, as confirmed by otoscopic examination. No animal had a history of loud noise exposure, ear trauma, or ototoxic drug treatment. All experimental procedures adhered to the guidelines of the Animal Advisory Committee at the Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

Forty rhesus monkeys aged 2 to 24 years were detected from Sichuan Primed Shines Bio-tech Co., Ltd. All experimental procedures adhered to the guidelines of the company and of the above Animal Advisory Committee and complied with laboratory animal ethics.

Anesthesia

Before anesthesia, the animals were fasted for more than 4 hours to prevent reflux from obstructing the airways. Marmosets were injected intramuscularly on the left hind leg with 0.12 mL atropine 10 minutes before inhalation anesthesia, to prevent gastroesophageal reflux. The animals were intramuscularly injected on the right hind leg with 0.1 mL ketamine. A custom-made mask was applied for oxygen and isoflurane inhalation to maintain good anesthesia. Rhesus monkeys were intramuscularly injected with 50 mg/mL ketamine to yield a dose per animal of 10 mg/kg.

During the anesthesia process, animals were treated with ECG and their blood oxygen was monitored to ensure their safety. Upon completion of testing, animals were kept warm and monitored until the effects of anesthesia disappeared, and were then returned to their home cages.

ABR

All recordings were performed in an electrically shielded and sound-attenuated chamber. ABR recordings were performed using 12 mm long, 27 gauge needle electrodes. Before insertion, the skin was carefully cleaned with alcohol. The recording electrode (+) was inserted subcutaneously at the intersection of the apex line of the two ears and the midline of the skull, the reference electrode (-) was inserted at the dorsal base on the stimulated side, and the ground electrode was subcutaneously inserted into the veutro part of the tail. Electrode impedance was measured after they were inserted into the skin, with impedance being below 1 kΩ for all recordings.

Auditory stimuli, consisting of tone bursts of duration 1.0 ms with 0.1 ms rise and fall times, were generated by an acoustic stimulator (RZ6, Tucker-Davis Technologies, Inc.) to induce estimated ABR peaks. These signals were delivered to a speaker (MF1 Multi-Function Speaker, Tucker-Davis Technologies, Inc.) inserted into the animal's ear and attached with a 5 cm-long tube (Tucker-Davis Technologies). Stimulus protocols for tone bursts were programmed using SigGenRZ software (Tucker-Davis Technologies, Inc.). The bioelectrical ABR signals recorded from the subdermal electrodes were transferred to a head stage (RA4LI, Tucker-Davis Technologies, Inc.) and forwarded to a preamplifier (RA4PA, Tucker-Davis Technologies, Inc.) with 20-fold amplification.

The responses to an average of 512 stimuli were amplified 100,000 times and filtered with high and low bandpasses of 100 Hz and 3 kHz, respectively. The stimulus output was reduced in 5 dB steps until the response was undetectable, with the first stimulus intensity that was unable to evoke any response defined as the threshold. Generally, wave II outputs became visible as stimulus intensities decreased. Tone bursts were fixed at 20 Hz. The effects of stimulus frequencies of 2 to 32 kHz on ABR latencies and amplitudes were evaluated at a stimulus intensity of 80 dB SPL. The latencies and amplitudes of the peaks were measured to the nearest 0.01 ms and the nearest 0.01 WV, respectively.

DPOAE

A probe microphone/loudspeaker system with two loudspeaker ports (ER10B+, Etymotic Research, Inc.) was inserted into the ear canal, and two loudspeakers (MF-1, Tucker-Davis Technologies, Inc.) were connected to the ER10B+ ports. Acoustic stimuli were delivered from an analog input/output (I/O) device (RZ6, Tucker-Davis Technologies, Inc.), through an electrostatic speaker driver (ED-1, Tucker-Davis Technologies, Inc.), to the MF-1 speakers. Each primary tone was delivered through a separate loudspeaker to avoid the generation of intermodulatory sounds, with responses recorded in an electrically shielded and sound-attenuated chamber. No spurious intermodulation products generated by the electronics or by the acoustics of the canal were detectable at the DPOAE frequencies assessed in these recordings. The data-acquisition module was connected to a personal computer (WS-8 workstation, Tucker-Davis Technologies, Inc.).

The sound stimulus consisted of simultaneous permanent pure tones at two different frequencies (f2/f1 ratio = 1.22), decreasing from 80 dB to 10 dB (L1 = L2) in 5 dB steps. DPOAEs were measured at six frequencies: 2, 4, 8, 16, 22, and 32 kHz. The DPOAE signals were displayed using TDT software.

Data analysis

Data were analyzed by analysis of variance (ANOVA), with statistical significance defined as P<0.05. Regression analyses were conducted hierarchically, with age (as a continuous variable) entered to account for between-subject variability, where R-squared equals the coefficient of determination.

For ABRs, the peak amplitude of each wave was defined as the average of the evoked potentials from the summit of the peak to the lowest points of the nearest troughs on the two sides. The peak latency for each peak was defined as the time from stimulus onset to the summit of the peak. Results of tests in which no observable waveform was present at any level for a tone stimulus were excluded from analysis.

For DPOAEs, the amplitude of each frequency at 80 dB SPL was defined as the numerical measure of sound level produced at a frequency of 2f1-f2 compared with background noise. Values below zero were scored as zero. The hearing threshold at each frequency was defined as the last detectable amplitude produced in response to the acoustic stimulation. Thresholds were determined as 80 dB SPL if DPOAE could not be elicited.

Immunohistochemistry

For the marmosets that died a natural death during the study period, the temporal bone region was dissected immediately after death and fixed 24 hours with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2). The tissue samples were transferred to 0.12 M EDTA for decalcification. EDTA was refreshed weekly for 2 weeks, and decalcified tissue was trimmed at each change of EDTA solution.

Decalcified cochleae were dissected into quarter turns, and the pieces were incubated in blocking reagent (8% normal donkey serum, 0.05% Triton X-100 in PBS) for 1 h at room temperature. The pieces were subsequently incubated overnight at room temperature with 1:300 anti-MYO7A primary antibody (Proteus Biosciences), 1:300 anti-TUJ1 primary antibody (BioLegend, 801202), 1:300 anti-NF-H primary antibody (Millipore, AB5539), 1:100 anti- Cleaved Caspase-3 (Cell Signaling Technology, 9661) or 1:300 anti-SOX2 primary antibody (R&D, AF2018). The tissue samples were subsequently washed three times with PBS and counterstained with 1:500 Alexa Fluor secondary antibodies (Invitrogen) for 1 hour, including donkey anti-rabbit Alexa Fluor 546 (A-21123), donkey anti-mouse Alexa Fluor 647 (A-31571) and donkey anti-rabbit Alexa Fluor 488 (A-21206). Nuclei were simultaneously stained with 5 mg/ml of a 1:1000 dilution of DAPI (D3571, Invitrogen). Confocal images were acquired on a Leica TCS SP8 laser confocal microscope using a 20× glycerin-immersion lens and the images were processed with ImageJ software.

Cryo-embedding and sectioning of cochleae

Cochleae, fixed and decalcified as described above, were incubated in 30% sucrose dehydration solution for 24 h at room temperature. The cochleae were transferred into OCT (Sakura Finetek USA, CA90501) in parallel with the cochlear axis while avoiding air bubbles. The tissues were placed in a -80° C freezer overnight followed by final orientation and sectioning into 10 μm thick samples using a freezing microtome 400 (LEICA CM3050S).

The resultant slides were incubated in blocking reagent (8% normal donkey serum, 0.05% Triton X-100) for 1 h at room temperature. After washing, the sections were incubated overnight at room temperature with 1:300 dilutions of primary anti-TUJ1 antibody (Bio Legend, 801202), primary anti-NF-H antibody (Millipore, AB5539), primary anti-SOX2 antibody (R&D, AF2018), primary anti-MAP2 antibody (SYSY, 188011) and primary anti-GFAP antibody (Novus, NB300-141). The tissue samples were washed three times with PBS and incubated for 1 hour at room temperature with 1:500 dilutions of the Alexa Fluor conjugated secondary antibodies (Invitrogen), including donkey anti-rabbit Alexa Fluor 546 (A-21123), donkey anti-mouse Alexa Fluor 647 (A-31571) and donkey anti-rabbit Alexa Fluor 488 (A-21206). Nuclei were stained with 5 mg/ml of a 1:1000 dilution DAPI (D3571, Invitrogen). Confocal Images were acquired on a Leica TCS SP8 laser confocal microscope using 20× and 40× glycerin-immersion lenses, and all images were processed with ImageJ software.