ORIGINAL ARTICLE Physiological and Morphological Assessment of the Saccule in Guinea Pigs After Noise Exposure Wei-Chung Hsu, MD, PhD; Jung-Der Wang, MD, PhD; June-Horng Lue, PhD; An-Shiou Day, MD; Yi-Ho Young, MD Objective: To investigate whether the saccule exhibits threshold shifts recovered 2 and 4 days, respectively, af- temporary or permanent functional loss resembling ter short-term noise exposure, with an interval of 2 days threshold shifts in auditory brainstem response (ABR) earlier in the recovery of VEMPs than that of ABR thresh- of guinea pigs following noise exposure. olds. In contrast, in group B, 78% and 83% of the ears exhibited permanent VEMP loss and ABR threshold shifts, Design: Randomly bred guinea pigs were divided into respectively, 10 days following long-term noise expo- 3 groups: A (short-term noise exposure, 30 minutes, sure. In group C, all animals showed normal VEMPs and n=15), B (long-term noise exposure, 40 hours, n=9), and ABRs throughout the study period. Light and electron C (no noise exposure, n=5). microscopic studies confirmed that loss of VEMPs cor- related with saccular lesion. Setting: University hospital. Conclusions: The saccule can exhibit temporary or per- Main Outcome Measures: All animals underwent ves- manent functional loss resembling hearing threshold shifts tibular-evoked myogenic potential (VEMP) and ABR tests. in guinea pigs following noise exposure. Recovery of Chronological changes of VEMP and ABR responses fol- VEMP precedes restoration of hearing threshold after dam- lowing noise exposure were analyzed and compared. Af- age from short-term noise exposure. Conversely, perma- ter audiovestibular function testing, animals were killed for nent VEMP loss after long-term noise exposure may re- morphological study with light and electron microscopy. flect permanent hearing threshold shifts. Results: In group A, temporary VEMP loss and ABR Arch Otolaryngol Head Neck Surg. 2008;134(10):1099-1106 XTREME NOISE CAN CLEARLY noise damages the hair cells of the cochlea damage hair cells in the and may also affect the saccular macula. cochlea, leading to tempo- Recently, the vestibular-evoked myo- rary or permanent thresh- genic potential (VEMP) has been vali- old shifts in hearing.1 How- dated to originate from the saccule and is Eever, the effect of noise on the vestibular easily recorded via the contracting neck part remains poorly understood.2,3 De- muscles using loud sound stimulation in hu- 9-11 spite numerous documented cases of bal- mans and experimental animals. The use ance disorders from noise-induced hear- of experimental animals, such as guinea pigs, ing loss, vestibular symptoms resulting facilitates the study of the mechanism of sac- from acoustic trauma have not been stud- cular disorders by recording VEMPs and confirming cell damage by morphological ied thoroughly. Furthermore, excessive ex- 12 posure to very loud music may also affect assessment. Hence, this study investi- gated whether VEMPs exhibit temporary or vestibular function. However, imbalance permanent loss resembling threshold shifts in noise-exposed workers or music- Author Affiliations: of the auditory brainstem response (ABR) exposed young people has not been ap- Department of Otolaryngology, in guinea pigs following noise exposure by National Taiwan University proved for compensation by insurance correlating the physiological results with Hospital, Taipei (Drs Hsu, Day, boards, possibly because vestibular dys- morphological changes. and Young); and Institute of function often recovers spontaneously via Occupational Medicine and central compensation.4 Industrial Hygiene, College of Phylogenically, the saccule in the lower METHODS Public Health (Drs Hsu and species such as amphibians and fish can act Wang) and Department of Anatomy and Cell Biology, as an acoustic receptor. Hence, intense ANIMAL PREPARATION College of Medicine (Dr Lue), sound and vibration may produce vestibu- National Taiwan University, lar reflexes, while the vestibular fibers can Randomly bred Hartley-strain guinea pigs Taipei. also respond to sound.5-8 Restated, loud weighing 250 to 300 g were housed at a mean (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 134 (NO. 10), OCT 2008 WWW.ARCHOTO.COM 1099 ©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 (SD) temperature of 23°C (2°C) and humidity of 55% (5%) and injection. Active and reference needle electrodes were in- fed a solid diet and tap water ad libitum. Fifteen animals re- serted in the vertex and ipsilateral retroauricular region, re- ceiving 30 minutes of continuous exposure to loud noise were spectively, while a ground electrode was placed in the neck of classified as group A (short-term noise exposure). Group B con- the animal. Click stimuli (duration, 0.1 ms) were delivered via sisted of 15 animals subjected to continuous noise exposure a plastic tube inserted into the ear canal to record the ABR for 40 hours (long-term noise exposure). After excluding 6 ani- (Smart EP2), monaurally. The repetition rate was 20/s, with a mals with otitis media during the study period, group B com- mean of 400 sweeps. The stimulus intensity began from 100-dB prised 9 animals. Two noise exposure levels were chosen be- peak equivalent SPL, followed by 10-dB step decrements until cause the 30-minute exposure would only produce temporary waveforms I, III, and V disappeared, thus determining the ABR threshold shifts, while 40 hours of exposure was guaranteed threshold. to produce permanent threshold shifts. Another 5 animals with no noise exposure (group C) served as a control group. MORPHOLOGICAL ASSESSMENT All animals in group A underwent ABR and VEMP tests dur- ing the pre–noise exposure period and after noise exposure on Following deep anesthesia with intraperitoneal injection of pen- days 0, 1, 2, 3, 4, and 7. For animals in group B, ABR and VEMP tobarbital sodium (50 mg/kg), the animals were transcardially tests were performed during the pre–noise exposure period and perfused with isotonic sodium chloride solution, followed by after noise exposure on days 0, 3, 7, 10 and 30. Throughout a fixative containing glutaraldehyde, 2.5%, in 0.1M phosphate the study, each animal in group C underwent the same tests as buffer at pH 7.4. After complete fixation, the animals were de- those in groups A and B. capitated, and the temporal bones were harvested and placed This study was approved by the institutional review board in the same fixative for 24 hours and decalcified with 10% EDTA and was conducted in accordance with the guideline for the care containing glutaraldehyde, 2.5%, at pH 7.4 for 1 week. Tissue and use of laboratory animals by the Animal Research Commit- blocks were cut horizontally into 200-µm thick slices with a tee, College of Medicine, National Taiwan University, Taipei. vibratome and postfixed in 1% osmium tetroxide for 1 hour. The sections were dehydrated in ascending ethanol solution, NOISE EXPOSURE infiltrated with propylene oxide, and finally embedded in Araldite-Epon mixture (Electron Microscopy Science; Fort Each animal in the 2 noise exposure groups was placed in a cage Washington; Pennsylvania). Semi-thin (1-µm) sections were and allowed to move freely. The noise level was measured with a cut by a Leica Ultracut E ultramicrotome (Leica, Vienna, Aus- sound level meter (model NA-24; Rion, Tokyo, Japan). Continu- tria) and stained with toluidine blue for light microscopic study. ous broadband white noise at a mean (SD) intensity of 115 (5)-dB For electron microscopic study (Hitachi 7100; Hitachi, Tokyo, sound pressure level (SPL) was produced by an audiometer (GSI Japan), ultrathin sections (60-80 nm) of the specimen were cut 61; Grason-Stadler Inc, Milford, New Hampshire) connected to and stained with 1% lead citrate. loud speakers on both sides of the cage. STATISTICAL ANAYLSIS VEMP TEST The abnormal percentages of VEMP results following short- or All animals underwent VEMP tests without general or local an- long-term noise exposure were compared by the McNemar test. esthesia. Needle electrodes were placed in dorsal neck muscle Chronological changes in ABR threshold following short- or on both sides at the level of the third cervical vertebral bone. A long-term noise exposure were compared against pre–noise ex- reference electrode was placed in the occipital area at the mid- posure threshold by the paired t test. Moreover, abnormal per- line, and electromyographic activity was recorded (Smart EP2; centages of VEMP and ABR results were compared by the Intelligent Hearing Systems, Miami, Florida). During the re- McNemar test. Trends in recovery of VEMP responses and ABR cording, each animal was immobilized in a prone position with thresholds were analyzed by Kaplan-Meier survival analysis. its head elevated and neck hyperextended. Electromyo- Finally, the log-rank test was used to compare recovery curves graphic signals were amplified and band-pass filtered between between VEMP and ABR. PϽ.05 was considered to be statis- 30 and 3000 Hz. Click stimuli (130-dB peak equivalent [pe] tically significant. SPL; duration, 0.1 milliseconds [ms]) were delivered via an ear- phone connected through a short tube inserted into the ear ca- RESULTS nal and stimulated in each ear separately. The stimulation rate was 5 beats/s, the analysis time for each response was 24 ms, and 200 responses were averaged for each run.11 BEHAVIOR CHANGE The positive-negative polarity of biphasic waveform termed AFTER NOISE EXPOSURE as waves I and II was measured based on their respective la- tencies of approximately 7 and 9 ms, respectively. Each ani- During noise exposure, most animals appeared agi- mal underwent serial tests with initial stimulus intensities of tated, while other animals kept immobile at 1 corner of 130-dB pe SPL, followed by 10-dB decrements until the wave- the cage, usually the corner farthest from the noise source.