Rapamycin Added to Diet in Late Mid-Life Delays Age-Related Hearing Loss in UMHET4 Mice

Abstract

Our previous study demonstrated rapamycin added to diet at 4 months of age had significantly less age-related outer hair cell loss in the basal half of the cochlea at 22 months of age compared to mice without rapamycin. The present study tested adding rapamycin to diet later in life, at 14 months of age, and added a longitudinal assessment of auditory brain stem response (ABR). The present study used UMHET4 mice, a 4 way cross in which all grandparental strains lack the Cdh23^753A allele that predisposes to early onset, progressive hearing loss. UMHET4 mice typically have normal hearing until 16-17 months, then exhibit threshold shifts at low frequencies/apical cochlea and later in more basal high frequency regions. ABR thresholds at 4, 12, 24, and 48 kHz were assessed at 12, 18, and 24 months of age and compared to baseline ABR thresholds acquired at 5 months of age to determine threshold shifts (TS). There was no TS at 12 months of age at any frequency tested. At 18 months of age mice with rapamycin added to diet at 14 months had a significantly lower mean TS at 4 and 12 kHz compared to mice on control diet with no significant difference at 24 and 48 kHz. At 24 months of age, the mean 4 kHz TS in rapamycin diet group was no longer significantly lower than the control diet group, while the 12 kHz mean remained significantly lower. Mean TS at 24 and 48 kHz in the rapamycin diet group became significantly lower than in the control diet group at 24 months. Hair cell counts at 24 months showed large loss in the apical half of most rapamycin and control diet mice cochleae with no significant difference between groups. There was only mild outer hair cell loss in the basal half of rapamycin and control diet mice cochleae with no significant difference between groups. The results show that a later life addition of rapamycin can decrease age-related hearing loss in the mouse model, however, it also suggests that this decrease is a delay/deceleration rather than a complete prevention.

Keywords: age-related hearing loss; aging; auditory; cochlea; deafness; rapamycin.

Figure 1

Comparison of mean auditory brain stem response threshold shifts (compared to 5 months of age) in the group with rapamycin added to diet at 14 months of age (rapa) vs. the group on control diet without rapamycin (control) at 12, 18, and 24 months of age at 4 kHz (A), 12 kHz (B), 24 kHz (C) and 48 kHz (D). Asterisks indicate significant differences. Please note differences between A, B, C, and D in scale bars for dB SPL on the “y” axis.

Figure 2

A scatter plot of the 4 kHz auditory brain stem response threshold shifts (compared to 5 months of age) at 12, 18, and 24 months of age in the control diet group (without rapamycin) and in the group with rapamycin added to diet at 14 months of age. There is little variability in either group at 12 months of age. At 18 months of age more variability is seen in the control diet group (control 18) while the rapamycin treatment group (RAPA18) remains more skewed toward little threshold shift. At 24 months of age there is less variability in the control group (control 24) now skewed toward large threshold shifts while the rapamycin treatment group (RAPA24) has more distribution across the range of threshold shifts.

Figure 3

Cytochleograms comparing the mean outer hair cell loss across the cochlea spiral at 24 months of age in mice from the group that had rapamycin added to diet at 14 months of age (red line) vs. the control diet group without rapamycin added to diet (blue line). Apical cochlea is to the left and base to the right, the transition from apical turn to basal turn is ~1.75 mm from the apex and the transition from basal turn to the hook is at ~3.9 mm from apex. There is large loss of outer hair cells in the apical third of the cochlea in both groups and minimal loss in the remaining cochlea (including hook) in both groups.

Figure 4

Representative photomicrographs from surface preparations of organ of Corti from a 24 months old Control Diet mouse (A) and a 24 months old mouse on the Rapamycin Diet (B) Both have outer hair cell loss in the third row, with scars marked by asterisks. Bar = 10 μ.