Wistar rats: a forgotten model of age-related hearing loss

Abstract

Age-related hearing loss (ARHL) is one of the most frequent sensory impairments in senescence and is a source of important socio-economic consequences. Understanding the pathological responses that occur in the central auditory pathway of patients who suffer from this disability is vital to improve its diagnosis and treatment. Therefore, the goal of this study was to characterize age-related modifications in auditory brainstem responses (ABR) and to determine whether these functional responses might be accompanied by an imbalance between excitation and inhibition in the cochlear nucleus of Wistar rats. To do so, ABR recordings at different frequencies and immunohistochemistry for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT) in the ventral cochlear nucleus (VCN) were performed in young, middle-aged and old male Wistar rats. The results demonstrate that there was a significant increase in the auditory thresholds, a significant decrease in the amplitudes and an increase in the latencies of the ABR waves as the age of the rat increased. Additionally, there were decreases in VGLUT1 and VGAT immunostaining in the VCN of older rats compared to younger rats. Therefore, the observed age-related decline in the magnitude of auditory evoked responses might be due in part to a reduction in markers of excitatory function; meanwhile, the concomitant reduction in both excitatory and inhibitory markers might reflect a common central alteration in animal models of ARLH. Together, these findings highlight the suitability of the Wistar rat as an excellent model to study ARHL.

Keywords: auditory brainstem response (ABR); cochlear nucleus; hearing loss; presbyacusis; rat model; vesicular transport proteins.

Figure 1

Line graphs illustrating the relationship between the auditory thresholds and the frequencies tested for each age group. Note that the mean values of the older groups rose as the age of the rats increased (A). Compared to the 6- to 8-month-old rats, the threshold shift in the 12- to 14-month-old rats ranged from approximately 24–33 dB and in the 18- to 20-month-old rats rose from approximately 30–46 dB (B). ^*p < 0.05; ^**p < 0.01.

Figure 2

Line graphs showing examples of ABR recordings of each age group at 80 dB SPL for all frequencies tested. In the three experimental groups, the recordings displayed a distinctive pattern characterized by 4–5 evoked waveforms after the stimulus onset. Despite the similarities between the groups, there was an apparent decrease in the wave amplitudes with age. The ABR recordings were compared between the 6- to 8-month-old rats (A) and both the 12- to 14-month-old (B) and the 18- to 20-month-old rats (C). Dashed lines indicate the stimulus onset. Stimulus intensity = 80 dB SPL.

Figure 3

Line graphs showing the wave amplitudes (μV) of each age group plotted as a function of frequency. In the 6- to 8-month-old rats, the mean amplitudes of all waves were higher compared to the 18- to 20-month-old group at all frequencies studied and higher than in the 12- to 14-month-old rats for several of the frequencies evaluated (A–E). Stimulus intensity = 80 dB SPL. ^*p < 0.05; ^**p < 0.01.

Figure 4

Bar graphs illustrating the percentage of variation in the wave amplitudes in older rats compared to 6- to 8-month-old rats. At all frequencies evaluated, compared to the 6- to 8-month-old group, the percent reduction in the wave amplitude grew as the age of the animals increased (A–E).

Figure 5

Line graphs illustrating the positive wave latency (ms) plotted as a function of the frequency. As shown, there was a significant effect of age on the positive wave latencies at the middle and high frequencies but not at the lower frequencies (A–E). This effect was more apparent for waves IV (D) and V (E). As indicated in the Methods section, 0.5 ms of acoustic transit time between the speaker's diaphragm and the rat's tympanic membrane was added to the latencies. Stimulus intensity = 80 dB SPL. ^*p < 0.05; ^**p < 0.01.

Figure 6

Line graphs illustrating the negative wave latency (ms) plotted as a function of the frequency. Similar to the positive latencies, a significant effect of age on the negative latencies of all waves was detected predominantly in the high and middle frequencies but not in the low frequencies (A–E). Additionally, the effect was more apparent in waves IV (D) and V (E). As indicated in the Methods section, 0.5 ms of acoustic transit time between the speaker's diaphragm and the rat's tympanic membrane was added to the latencies. Stimulus intensity = 80 dB SPL. ^*p < 0.05.

Figure 7

Line graphs showing the interpeak latency (ms) plotted as a function of the frequency. Evaluation of the interpeak latencies revealed significant differences at higher frequencies for both the positive and negative interpeak latencies between waves II and IV (C–D) and between waves I and IV (E–F), but not between waves I and II (A–B) among the three age groups. Stimulus intensity = 80 dB SPL. ^*p < 0.05.

Figure 8

Digitized images illustrating VGLUT1 immunoreactivity in the AVCN in 6- to 8-month-old (A,D,G), 12- to 14-month-old (B,E,H), and 18- to 20-month-old (C,F,I) Wistar rats. In the three experimental groups, VGLUT1 immunostaining (D–F) appeared mainly as large profiles (arrows in D–F) surrounding neurons in the CN (asterisks in D–F), as well as small endings in the neuropil. Immunopositive profiles in 6- to 8-month-old rats (D,G) appeared to be more abundant than in 12- to 14-month-old (E,H) and 18- to 20-month-old (F,I) rats. Bar graphs indicate the mean gray level of VGLUT1 immunostaining (J) and the immunostained area (K) in the AVCN. The mean gray level in the 18- to 20-month-old rats (J) and the immunostained area in both the 12- to 14-month-old rats and the 18- to 20-month-old rats were lower compared to the 6- to 8-month-old rats (K). The error bars indicate the standard errors of the mean. The square box in A indicates the approximate location of the high-magnification images illustrated in D–I. Scale bars represent 250 μm in C, 25 μm in F, and 20 μm in G. ^*p < 0.05; ^**p < 0.01.

Figure 9

Digitized images illustrating VGLUT1 immunostaining in the PVCN in 6- to 8-month-old (A,D,H), 12- to 14-month-old (B,E,I), and 18- to 20-month-old (C,F,G,J) rats. Similar to the AVCN, VGLUT1 immunostaining in the PVCN consisted of perisomatic profiles (arrows and asterisks in D–G) and puncta throughout the neuropil. There were also noticeable differences between the groups, such that immunostaining was more profuse in the 6- to 8-month-old rats (D,H) in comparison to the 12- to 14-month-old (E,I) and 18- to 20-month-old (F,G,J) rats. Bar graphs indicate the mean gray level (K) and the area (L) of VGLUT1 immunostaining. The mean gray levels in both the 12- to 14-month-old and the 18- to 20-month-old rats (K) and the immunostained area in both the 12- to 14-month-old and in the 18- to 20-month-old rats (L) were lower than in the 6- to 8-month-old rats. The error bars indicate the standard errors of the mean. The square box in A indicates the approximate location of the high-magnification images illustrated in D–J. Scale bars represent 250 μm in C, 25 μm in D, and 20 μm in H. ^*p < 0.05.

Figure 10

Digitized images illustrating VGAT immunostaining in the AVCN in 6- to 8-month-old (A–C), 12- to 14-month-old (D–F), and 18- to 20-month-old (G–I) rats. An apparent decrease in the immunostaining of large and small inhibitory synaptic endings was observed with age, with VGAT profiles (arrows and asterisks) distributed more abundantly in the 6- to 8-month-old rats (B,C) than in both the 12- to 14-month-old (E,F) and 18- to 20-month-old (H,I) rats. Bar graphs indicate the mean gray level (J) and the area (K) of VGAT immunostaining in the three age groups. Both the mean gray level and the immunostained area in the 18- to 20-month-old rats were significantly lower compared to both the 6- to 8-month-old rats and the 12- to 14-month-old (J,K) rats. The square box in A indicates the approximate location of the high-magnification images shown in B,C,E,F,H,I. Scale bars represent 250 μm in G, 25 μm in H, and 20 μm in I. ^*p < 0.05; ^**p < 0.01.

Figure 11

Digitized images illustrating VGAT immunostaining in the PVCN in 6- to 8-month-old (A–E), 12- to 14-month-old (F,H) and 18- to 20-month-old (I,L) rats. Similar to the AVCN, VGAT immunostaining was also more profuse in the 6- to 8-month-old (arrows and asterisks in B–E) rats than in both the 12- to 14-month-old (arrows and asterisks in G,H) and 18- to 20-month-old (arrows and asterisks in J–L) rats. Bar graphs indicate the mean gray level (M) and the immunostained area (N) of VGAT immunostaining. No differences between groups were detected in the mean gray level (M), but the immunostained area (N) in the 18- to 20-month-old rats was significantly lower than in both the 6- to 8-month-old and 12- to 14-month-old rats. The error bars indicate the standard errors of the mean. The square box in A indicates the approximate location of the high-magnification images illustrated in B–E, G–H, and J–L. Scale bars represent 250 μm in I, 25 μm in J, and 20 μm in L. ^*p < 0.05; ^**p < 0.01.