The FBN rat model of aging: investigation of ABR waveforms and ribbon synapse changes

Abstract

Age-related hearing loss is experienced by one-third of individuals aged 65 years and older and can be socially debilitating. Historically, there has been poor correlation between age-related threshold changes, loss of speech understanding, and loss of cochlear hair cells. We examined changes in ribbon synapse number at four different ages in Fisher Brown Norway rats, an extensively studied rat model of aging. In contrast to previous work in mice/Wistar rats, we found minimal ribbon synapse loss before 20 months, with significant differences in 24- and 28-month-old rats at 4 kHz. Significant outer HC loss was observed at 24 and 28 months in low- to mid-frequency regions. Age-related reductions in auditory brainstem response wave I amplitude and increases in threshold were strongly correlated with ribbon synapse loss. Wave V/I ratios increased across age for click, 2, 4, and 24 kHz. Together, we find that ribbon synapses in the Fisher Brown Norway rat cochlea show resistance to aging until ∼60% of their life span, suggesting species/strain differences may underpin decreased peripheral input into the aging central processor.

Keywords: Age-related hearing loss; Auditory brainstem response; Fisher Brown Norway rat; Ribbon synapses.

Figure 1

ABR thresholds of FBN rats increased with age. All groups showed lowest threshold at 12 kHz. Significant differences were detected between groups for click (two-way ANOVA followed by Tukey’s posthoc test, p < 0.001) and all frequencies tested (p < 0.001 for 2 kHz, 4 kHz, and 12 kHz; p < 0.01 for 24 kHz). N = 9 (4–6m), 9 (20m), 8 (24m), 7(28m). Data are presented as mean ± SEM. **p < 0.01; ***p < 0.01. Detailed between group comparisons (Table 1).

Figure 2

ABR waveform amplitudes of FBN rats at four ages. (A) Sample ABR waveforms elicited by 12 kHz at 80 dB from a young (4–6m) and an aged (28m) rat. Decreased amplitude of all waves was detected when comparing waveforms between young and aged rats. Arrows indicated the peak (P) and trough (N) measurements of wave I (P1, N1) and V (P5, N5) for the young animal waveform. (B, C) Wave I and V amplitudes from click, 2 kHz, 4 kHz, 12 kHz, and 24 kHz for the four age groups (no data from 28m at 2 kHz presented due to the high threshold of the aged rats), respectively. There were significant age-related differences in wave I amplitudes for click (p < 0.001), 2 kHz (p < 0.05), 4 kHz (p < 0.05), 12 kHz (p < 0.01), and 24 kHz (p < 0.05). Wave V amplitude remained relatively less changed except for the click evoked response (p < 0.01). There was a p = 0.05 decrease at 12 kHz among four groups. (D) Wave V/I amplitude ratio for click, 2 kHz, 4 kHz, 12 kHz, and 24 kHz from the four age groups. Wave V/I ratio for 12 kHz showed no age-related change. There were significant age-related increases in the wave V/I ratios for click, 2 kHz, 4 kHz, and 24 kHz (p < 0.05). Data are presented as mean ± SEM. Two-way ANOVA followed by a Tukey’s posthoc test, *p < 0.05; **p < 0.01; ***p < 0.01. N = 9 (4–6m), 9 (20m), 8 (24m), 7(28m). Detailed group comparisons (Tables 2–4).

Figure 3

Cytocochleogram of FBN rat cochleae at four ages. Representative maximum projection confocal images show a well-organized organ of Corti with three rows of OHCs and one row of IHCs at (A) 4–6m, (B) 20m, (C) 24m, and (D) 28m of age. HCs are labeled by myosin VIIa (myo7a, green). (E) No significant change in IHC number was observed at any age or frequency distribution along the cochlea [two-way ANOVA F (9, 45) = 1.454]. (F) Significant age-related changes in OHC number was detected at 24m in the 2 kHz region and at 28m in the 2 kHz, 4 kHz, and 12 kHz regions. Data are presented as mean ± SEM. N = 5 for 4–6m; 4 for 20m; 5 for 24m and 5 for 28m. Two-way ANOVA [F (9, 45) = 6.698] followed by a Tukey’s posthoc test,*p < 0.05; **p < 0.01, ****p < 0.0001.

Figure 4

IHC-SGN synapse changes at 4 kHz in the FBN rat cochlea at four ages. Representative maximum projection confocal images showing age-related changes in synapses located on IHCs [myosin VIIa, Myo7a (blue)]. Presynaptic regions are labeled by Ctbp2 (magenta), and postsynaptic glutamate receptors are labeled by GluR2 (green). (A) Merged image showing seven IHCs from the 4 kHz region in a 4–6m rat. Box indicates synapses in one IHC, with the higher magnification image shown in (B). Representative high magnification images of IHC-SGN synapse from the 4 kHz region of 20m (C), 24m (D), and 28m (E) FBN rat cochleae. Arrows indicate orphan synapses. Scale bar in A = 10 μm and B = 5 μm.

Figure 5

Quantification of IHC-SGN synapse number in FBN rat cochleae at four ages. Number of IHC-SGN synapses (A) and orphan synapses (B) per IHC at 2, 4, 12, and 24 kHz in FBN rats at four ages. There was a significant reduction in IHC-SGN synapses at 24m (p < 0.001) and 28m at 4 kHz (p < 0.001). In the 2 kHz region, there were significantly more orphan synapses in 28m samples compared to 4–6m (p < 0.05) samples. In the 4 kHz region, there were significantly more orphan synapses in 28m samples compared to 4–6m (p < 0.01), 20m (p < 0.001), and 24m (p < 0.05) samples. (C) The number of IHC-SGN synapses were plotted against the percentage of lifespan. Significant synapse loss was observed starting at 75% of the FBN lifespan at 4 kHz. Data are presented as mean ± SEM. N = 5 for 4–6m; 4 for 20m; 5 for 24m and 5 for 28m. Two-way ANOVA [F (9, 45) = 0.8485 for A and F (9, 45) = 1.190 for B] followed by a Tukey’s posthoc test,*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

ABR wave I amplitude significantly correlates with wave I threshold and the number of IHC-SGN synapses regardless of age. (A) Negative correlations were found between wave I amplitude and ABR threshold with the highest correlation at 4 kHz (p < 0.05 for 2 kHz; p < 0.001 for 4 kHz, 12 kHz, 24 kHz and click). (B) Positive correlations were detected between wave I amplitude and the number of IHC-SGN synapses for 4 kHz (R²= 0.54, p < 0.001), 24 kHz (R²= 0.32, p < 0.05), but not 2 kHz (R²= 0.15, p = 0.21). Data were analyzed using a Pearson correlation coefficient test. N = 12 for 2 kHz, 18 for 4 kHz, 19 for 12 kHz, and 19 for 24 kHz. (Table 5).

Figure 7

ABR wave V/I ratio significantly correlates with OHC and IHC-SGN synapses. Correlation between wave V/I ratio and OHC numbers or IHC-SGN synapses were calculated at 2, 4, 12, and 24 kHz regardless of age. Significant negative correlations were found between wave V/I ratio and OHC numbers at 4 kHz (A) (p < 0.01, Pearson correlation coefficient test, N = 17); and between wave V/I ratio and IHC-SGN synapse number at 24 kHz (B) (p < 0.001, Pearson correlation coefficient test, N = 19).