Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation

Abstract

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this "toughening" effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with "toughening" and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

Keywords: Wistar rats; calcium; cochlear nucleus; conditioning; priming; toughening.

Figure 1

(A) Wistar rats were exposed to a noise-induced toughening protocol consisting of 1 h continuous white noise at 118 dB SPL in four different sessions, with a recovery time of 72 h between sessions. The ABR recordings were performed before the noise exposure and then at 24 (1S1H24H) and 72 h (1S1H72H) after the first session and at 24 (4S1H24H) and 72 h (4S1H72H) after the fourth session of the noise-induced toughening protocol. (B) Line graphs illustrating the relationship between the auditory thresholds and the frequencies evaluated in control and experimental groups. Note that the mean values in 1S1H24H rats were increased at all frequencies compared to control and the other experimental groups. Values in 4S1H24H animals were slightly increased mainly in the higher frequencies when compared to control and experimental rats. (C) In comparison to the control condition, the largest threshold shift was observed at 24 h after the first session and ranged from 28.93 to 44.96 dB. However, by 24 h after the fourth session the threshold shift varied from 8.35 to 16.34 dB. Color arrows indicate the time point at which the ABRs were performed and black dashed arrows show the corresponding sessions of the noise-induced toughening protocol.

Figure 2

Line graphs showing examples of ABR recordings in control and experimental rats at 80 dB SPL for all frequencies evaluated. (A) In the control group, the recordings displayed a distinctive pattern characterized by 4–5 evoked waveforms after the stimulus onset. (B) By 24 h after the first session of the noise-induced toughening protocol there was a significant decrease in the amplitude of all waveforms in the ABR. (C) The amplitudes apparently returned to values close to the normal condition in 1S1H72H animals. (D) In 4S1H24H rats, although there was a slightly decrease in the waves amplitude, it was not as evident as in 1S1H24H rats. (E) The recordings in the 4S1H72H condition were similar to those observed in control. Dashed lines indicate the stimulus onset. Stimulus intensity = 80 dB SPL.

Figure 3

Line graphs showing the amplitudes (in μV) and contrast index of waves I, II, and IV plotted as a function of frequencies in control and experimental animals. Before sound stimulation, wave amplitudes were larger in the lower frequencies and smaller at medium and higher frequencies, being wave II the largest of all (A–C). However, by 24 h after the first session there was a significant decrease in all waves amplitudes (A–C), with contrast indexes below −0.2 in all waves and at all frequencies (D–F). The amplitudes (A–C) as well as the contrast index (D–F) were recovered by the fourth session. Stimulus intensity = 80 dB SPL. (1) Control; (2) 1S1H24H; (3) 1S1H72H; (4) 4S1H24H; (5) 4S1H72H. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 4

Line graphs illustrating the latencies of the positive and negative peaks (ms) of waves I, II, and IV plotted as a function of the frequencies in control and experimental conditions. In 1S1H24H rats, there were significantly longer positive and negative peak latencies at the middle and high frequencies (A–F). These values returned to normal at the fourth session of noise exposure (A–F). 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 included in the latencies. Stimulus intensity = 80 dB SPL. (1) Control; (2) 1S1H24H; (3) 1S1H72H; (4) 4S1H24H; (5) 4S1H72H. ^*p < 0.05; ^**p < 0.01.

Figure 5

Line graphs showing the positives and negatives interpeak intervals (ms) of waves I, II, and IV plotted as a function of the frequencies in control and experimental animals. Whereas no differences were found between waves I and II (A,D), significantly longer positives and negatives interpeak intervals at 32 kHz were observed between waves II and IV (B,E) and between waves I and IV (C,F) by 24 h after the first session. These differences were no longer present by the fourth session of noise overstimulation. Stimulus intensity = 80 dB SPL. (1) Control; (2) 1S1H24H; (3) 1S1H72H; (4) 4S1H24H; (5) 4S1H72H. ^*p < 0.05.

Figure 6

Digitized images illustrating CR immunoreactivity in the spiral ganglion (A–J) and the spiral ligament (K–P) in control and experimental Wistar rats. Before noise overstimulation, CR immunostaining in the spiral ganglion was characterized by dark immunopositive somata and fibers that were lightly stained throughout the ganglion (A,F). Meanwhile, in the spiral ligament, the staining was present in type I fibrocytes that were moderately stained and distributed in the dorsolateral area (arrows and inset in L). Following the noise-induced toughening protocol, there was an increase in the immunostaining in spiral ganglion neurons and neuropil in 1S1H24H (B,G) rats that was still present at 1S1H72H (C,H), 4S1H24H (D,I), and 4S1H72H (E,J) animals relative to the control condition. Similar increases were also observed in the spiral ligament (arrows and inset in M–P) when compared to the control condition (arrows and inset in K,L). Bar graphs indicate the mean gray levels of CR immunostaining and its corresponding percentage of variation in the spiral ganglion (Q,R) and spiral ligament (S,T) in experimental and control rats. The square box in (K) indicates the approximate location of the high-magnification images illustrated in (L–P). Scale bars represent 100 μm in (K), 50 μm in (E), and 25 μm in (J,P). SG, spiral ganglion; SL, spiral ligament; SV, stria vascularis.

Figure 7

Digitized images illustrating nNOS immunostaining in the spiral ganglion (A–J) and the spiral ligament (K–P) in control and experimental Wistar rats. Before sound conditioning, nNOS immunostaining in spiral ganglion was located within neurons that were lightly immunostained (A,F), whereas in the spiral ligament, the staining was weak and mainly distributed in the region where types I and III fibrocytes were found (arrows in L; also see L1-L2). In the 1S1H24H condition, nNOS levels in the spiral ganglion were upregulated (B,G) and persisted elevated in 1S1H72H (C,H), 4S1H24H (D,I) and 4S1H72H (E,J) groups when compared to control rats. A similar upregulation was also observed in the spiral ligament (arrows in K–P and arrows in K1–P2). Bar graphs indicate the mean gray levels of nNOS immunostaining in the spiral ganglion (Q) and the spiral ligament (S) and their corresponding percentage of variation (R,T). The squares boxes in (K) indicate the approximate location of the high-magnification images illustrated in (L–P). Scale bars represent 100 μm in (K), 50 μm in (E), and 25 μm in (J) and (P2). SG, spiral ganglion; SL, spiral ligament; SV, stria vascularis.

Figure 8

Digitized images illustrating CR immunostaining in the AVCN in control and experimental Wistar rats. At 24 h after the first session of sound conditioning, CR levels in both neurons and neuropil were increased (arrows in C), and continued elevated in 1S1H72H (arrows in D), 4S1H24H (arrows in E), and 4S1H72H (arrows in F) conditions when compared to control rats (arrows in B). Bar graphs show the mean gray level of CR immunostaining (G) and its percentage of variation (H) in the experimental conditions relative to control. The square box in (A) indicates the approximate location of the high-magnification images shown in (B–F). Scale bars represent 250 μm in (A), and 50 μm in (C).

Figure 9

Digitized images illustrating CR immunostaining in the PVCN in control and experimental Wistar rats. Similar to that in the AVCN, CR levels in both neurons and neuropil were augmented in all the experimental conditions (arrows in C–F) when compared to control rats (arrows in B). Bar graphs show the mean gray level of CR immunostaining (G) and the percentage of variation (H) in the experimental conditions with respect to control. The square box in (A) indicates the approximate location of the high-magnification images shown in (B–F). Scale bars represent 250 μm in (A), and 50 μm in (C).

Figure 10

Digitized images illustrating nNOS immunostaining in the AVCN in control and experimental Wistar rats. nNOS-immunostaining was increased by 24 h after the first session (arrows in C), and diminished in 1S1H72H (arrows in D), 4S1H24H (arrows in E), and 4S1H72H (arrows in F) conditions to reach normal values (arrows in B). Bar graphs indicate the mean gray level of nNOS immunostaining in the AVCN (G) and its percentage of variation (H) in the experimental conditions relative to control. The square box in (A) indicates the approximate location of the high-magnification images shown in (B,C,E,F). Scale bars represent 250 μm in (A), and 50 μm in (F).

Figure 11

Digitized images illustrating nNOS immunostaining in the PVCN in control and experimental Wistar rats. Similar to that in the AVCN, nNOS immunostaining in the 1S1H24H condition was increased (arrows in C) when compared to the control condition. Although nNOS levels decreased in 1S1H72H (arrows in D) rats, they increased slightly in 4S1H24H (arrows in E) and 4S1H72H (arrows in F) animals when compared to the control condition (arrows in B). Bar graphs indicate the mean gray level of nNOS immunostaining (G) and the percentage of variation (H) in the experimental conditions with respect to control. The square box in A indicates the approximate location of the high-magnification images shown in (B,C,E,F). Scale bars represent 250 μm in (A), and 50 μm in (F).

Figure 12

Digitized high magnification images depicting SYN immunostaining in the AVCN in control and noise-exposed rats. The staining consisted of punctate deposits in the neuropil and perisomatic profiles that were seen surrounding unstained cochlear nucleus neurons (asterisks in A,D,G,J) immunostained either with CR (asterisks in B,E,H,K) or with NOS (asterisks in C,F,I,L). Although there were no differences in the distribution pattern of SYN among groups, significant increases in CR and NOS staining were evident in 1S1H24H (E,F) rats when compared to control (B,C). Scale bars represent 25 μm in (J,L).

Figure 13

Digitized high magnification images depicting SYN immunostaining in the PVCN in control and noise-exposed rats. Stained endings mainly distributed around either CR or NOS immunostained cochlear nucleus neurons (asterisks in A–N). The distribution pattern of SYN immunostaining was not different among groups. Note that there was an upregulation in NOS and CR staining by 24 h after the first session (D–F). Scale bars represent 25 μm in (G,N).