Thresholds are shown 1 hour after the first exposure to acoustic overstimulation events (AOSEs) on day 1, days 4 and 8, day 8 1 hour after the second exposure to AOSEs, and days 11 and 15. Group 1 received an intraperitoneal injection of caffeine, 25 mg/kg, each study day; group 2, AOSE exposure on days 1 and 8; and group 3, both protocols. We used the Mann-Whitney test to compare groups 2 and 3 at each interval. A, B, and D, Impairment in recovery in group 3 was statistically significant on days 4, 8, 11, and 15. C, Impairment in recovery was not statistically different at any time. SPL indicates sound pressure level.
aIndicates 1 hour after exposure to an AOSE.
The image is from a guinea pig from group 3 (intraperitoneal injection of caffeine, 25 mg/kg, each study day and exposure to acoustic overstimulation events on days 1 and 8) and demonstrates a grade 2 damage to the outer hair cells (count of <50%) (hematoxylin-eosin stain, original magnification ×200).
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Zawawi F, Bezdjian A, Mujica-Mota M, Rappaport J, Daniel SJ. Association of Caffeine and Hearing Recovery After Acoustic Overstimulation Events in a Guinea Pig Model. JAMA Otolaryngol Head Neck Surg. 2016;142(4):383–388. doi:10.1001/jamaoto.2015.3938
Noise-induced hearing loss is an increasingly worrisome problem. Although caffeine intake is common in people involved in noise-related environments, the effect of caffeine on the recovery of hearing after a temporary threshold shift requires further understanding.
To determine whether caffeine impairs hearing recovery in a guinea pig model exposed to acoustic overstimulation.
Design, Setting, and Subjects
This experiment at the McGill University Auditory Sciences Laboratory used 24 female albino guinea pigs (age, 6 months; weight, 500-600 g) divided randomly into 3 groups of 8 animals each. Group 1 was exposed to caffeine; group 2, acoustic overstimulation events (AOSEs); and group 3, both. Data were collected from July 1, 2013, to March 30, 2014, and analyzed from April 1 to August 1, 2014.
Daily caffeine dose for groups 1 and 3 consisted of 25 mg/kg administered intraperitoneally for 15 days. The AOSEs were administered on days 1 and 8 and consisted of 1 hour of 110-dB pure-tone sound.
Main Outcomes and Measures
Serial auditory brainstem response (ABR) tests to determine the audiological threshold shift and recovery were obtained at baseline and on days 1 (1 hour after the first AOSE), 4, 8 (before and 1 hour after the second AOSE), 11, and 15. Scanning electron and light microscopy of the cochleas were performed to determine morphologic changes.
The day 1 post-AOSE measurement resulted in a similar threshold shift in all animals in groups 2 and 3 at all frequencies tested (8, 16, 20, and 25 kHz). The maximum threshold shift was at 16 kHz, with a mean of 66.12 dB. By day 8, the threshold shift in group 2 recovered completely at all frequencies except 20 kHz, where a mean threshold shift of 20.63 dB of sound pressure level (SPL) was present. Hearing impairment in group 3 persisted in 8-, 16-, and 25-kHz frequencies with thresholds of 21.88, 28.13, and 26.25 dB SPL, respectively (P = .001). After a second AOSE at day 8, similar threshold shift and outcome were recorded on day 15 compared with day 8, with a mean threshold shift at 20 kHz of 29.38 dB SPL in group 2 and mean threshold shifts at 8, 16, 20, and 25 kHz of 29.38, 35.63, 40.63, and 38.75 dB SPL, respectively, in group 3. The difference in ABR threshold recovery was in concordance with scanning electronic and light microscopy findings for each group.
Conclusions and Relevance
A daily dose of caffeine was found to impair the recovery of hearing after an AOSE.
Noise-induced hearing loss is a common problem among young adults and adolescents. Exposure to loud noise is present in various settings, which can be recreational or work related.1 The mechanisms of this type of hearing loss are auditory hair cell trauma, promotion of a hypoxic environment with strial atrophy, and spiral ganglion neuronal degeneration.2,3
The most ingested psychoactive substance is caffeine,4 which is found in common beverages such as soda, energy drinks, coffee, and tea. Caffeine has been found to increase the release of dopamine, serotonin, and norepinephrine.5 Caffeine also has behavioral effects by increasing arousal and decreasing fatigue. Nevertheless, caffeine has a long list of adverse effects, including insomnia, tremors, seizures, and anxiety.6
Teenagers and adolescents consume caffeine in alarming amounts. Pollak and Bright7 reported a caffeine intake of as much as 800 mg/d in eighth and ninth grade students. When these levels of intake are compared with those of adults, a recent review of the association between caffeine and urinary incontinence noted that some patients consume more than 2.4 L of caffeine-containing drinks per day.8
Owing to the increased consumption of caffeine, interest in the association of caffeine with hearing loss has been growing. These studies are clinically relevant and based on the environments and activities where regular caffeine consumption occurs in noisy leisure (eg, concerts and loud music clubs) or occupational (eg, machine works and aviation industry) settings.
A recent pilot study9 has demonstrated that caffeine-treated animals experience a delay in hearing recovery after exposure to loud noise. Caffeine may induce such a phenomenon through many possible mechanisms, but none of them is well understood. The objective of this study was to determine the effect of daily caffeine intake on hearing recovery after acoustic overstimulation events (AOSEs) in a guinea pig model. We hypothesized that daily intake of caffeine impairs the recovery of hearing after an AOSE.
Question: What is the effect of caffeine on hearing recovery after an acoustic overstimulation event (AOSE)?
Findings: In this guinea pig model, animals receiving daily intraperitoneal caffeine doses had impaired recovery of hearing in 3 different frequencies after exposure to pure-tone AOSEs. These findings were statistically significant compared with those of animals that did not receive caffeine.
Meaning: Caffeine exposure was found to impair the recovery of hearing after an AOSE.
This study was performed from July 1, 2013, to March 30, 2014. We used 24 female albino guinea pigs (age, 6 months; weight, 500-600 g). The animals were housed at 22°C (±4°C) with a light-dark cycle of 12 hours. They had access to food and water ad libitum. This study was approved by the ethics review board of McGill University and the McGill University animal care committee.
The guinea pigs were divided randomly into 3 groups. Group 1 (n = 8) received a daily intraperitoneal injection of caffeine, 25 mg/kg, for 15 days (control condition). Group 2 (n = 8) received 2 AOSEs on days 1 and 8. Group 3 (n = 8) received a daily intraperitoneal injection of caffeine, 25 mg/kg, for 15 days and 2 AOSEs on days 1 and 8. The randomization process was performed by one of us (M.M.-M.) who was not involved in the auditory brainstem response (ABR) testing.
The choice of caffeine dose was based on the literature review8 and previous research conducted in our laboratory.10 In the literature, a dose of greater than 25 mg/kg/d was considered an excessive dose of caffeine that resulted in significant physiologic changes, including urinary incontinence. The weight and behavior of the animals were recorded every day throughout the experiment. Each dose was calculated based on the daily weight (25 mg/kg/d) of the animal and administered each day of the study from 9 to 10 am.
The study adopted the method for AOSEs used in previously published studies.9,11,12 In a soundproof room, the guinea pigs were anesthetized using ketamine (50 mg/kg) and xylazine (1 mg/kg). Each AOSE consisted of a continuous 6-kHz pure-tone noise produced by a generator (Intelligent Hearing Systems) and amplified (D-75A amplifier; Crown Audio Inc). The tone was then projected using 2 loudspeakers at a distance of 5 cm from the animal’s head in a free field in the soundproof room. A calibrated sound level meter (Bruel & Kjaer) was used to monitor the sound pressure level (SPL). The sound was amplified to 110 dB SPL and sustained for 1 hour.9,11,12 Each animal in groups 2 and 3 (AOSEs and AOSEs + caffeine) were exposed to AOSEs on days 1 and 8 of the experiment.
Under general anesthesia using 5% inhaled isoflurane for induction and 2% inhaled isoflurane for maintenance, ABR was performed. Animals with abnormal anatomy of the ear were excluded. Before measurement, the ABR machine was calibrated to the industrial standard provided by the company in a similar manner to the pilot study.9 The ABRs were measured at the following intervals: baseline (before the start of the experiment) and on days 1 (1 hour after the first AOSE), 4, 8 (before and 1 hour after the second AOSE), 11, and 15.
The testing was performed only on the right ear of all animals using an evoked potentials system (SmartEP; Intelligent Hearing System). Electrodes were placed subdermally on the vertex (reference), the contralateral ear (ground), and the tested right ear (active). Frequencies tested included 8-, 16-, 20-, and 25-kHz tone-burst stimuli.9
The stimuli presented to the right ear were initially presented at 100 dB SPL and then decreased to 5 dB SPL. The responses were filtered and amplified, and the mean of 1600 sweeps was calculated. The hearing threshold was considered the lowest intensity at which waves III and V were obtained. A threshold shift was calculated based on the difference between the threshold at the specific time point and the baseline at that specific frequency.
On day 15 of the experiment after the final ABR test, all animals were killed humanely. Four randomly selected animals from each group had their right cochleas removed, fixed with 2.5% glutaraldehyde for 2 hours, and soaked in a 0.1M phosphate-buffered saline solution for 24 hours at 4°C. After fixation, the cochleas were treated with osmium tetroxide for 90 minutes and dehydrated in 70% ethanol. Subsequently, the cochlea was drilled and the organ of Corti was dissected. The sample then was dehydrated in 100% ethanol, and scanning electron microscopy was performed. To quantify the effect on the outer hair cells (OHC), we adopted a modified version of the 4-grade scale of Saito et al,13 where N indicates normal; 1, 10% to 50% OHC loss or damage; 2, OHC count of less than 50%; and 3, cuticular plate rupture and missing hair cells. Results were obtained for the apical, middle, and basal turns.
The other 4 animals in the each group had their right cochleas extracted immediately after they were killed humanely. Fixation was performed with 10% formalin for 48 hours. After 3 weeks of decalcification, the cochleas were dehydrated using 50% to 100% ethanol and cut into 5-μm sections. Hematoxylin-eosin staining was performed, and the sections were mounted for light microscopy. The turns of the cochlea were then captured at 200 × magnification using a digital camera (AxioCam MR3; Carl Zeiss).
Data were analyzed from April 1 to August 1, 2014. Initially, analysis of group 1 (caffeine only) was performed during the time from baseline to day 14 to determine the effect of caffeine on hearing. After calculating the threshold shift for each frequency at each time frame, the mean values of each group were analyzed with Kruskal-Wallis analysis of variance (ANOVA) to determine significance among all 3 groups. Next, we used the Mann-Whitney test to determine differences between groups 2 and 3. The results were considered statistically significant at P ≤ .05. Analysis was performed using SPSS software (version 23.0; SPSS, Inc).
The baseline ABR threshold analysis showed no difference among any of the 3 groups (P > .05). Daily intraperitoneal injection of caffeine was not found to cause the ABR threshold shift during the 15 days of injections. On day 1, 1 hour after an AOSE, we noted a threshold shift in groups 2 and 3. This shift was not different between the groups (P > .05, Kruskal-Wallis analysis of variance). The maximum shift was observed on the 16-kHz frequency (66.12 dB SPL).
Group 2 (AOSEs only) ABRs showed complete recovery of the threshold shift by day 8 at all frequencies except 20 kHz, where a mean threshold shift of 20.63 dB SPL was present. Similar results were obtained after the second AOSE. At day 15, the ABR of group 2 demonstrated hearing recovery at all frequencies except 20 kHz, where a mean threshold shift of 29.38 dB SPL persisted.
In group 3 (AOSEs + caffeine), threshold shift recovery was impaired at days 4 and 8 in all frequencies. The mean threshold shifts on day 8 at 8, 16, 20, and 25 kHz were 21.88, 28.13, 36.25, and 26.25 dB SPL, respectively. Kruskal-Wallis ANOVA showed that these results were statistically significant compared with the baseline values (P < .05). These results were then reproduced at the second week of the testing. At day 15, the mean threshold shifts at 8, 16, 20, and 25 kHz were 29.38, 35.63, 40.63, and 38.75 dB SPL, respectively. These results were statistically different from baseline levels (P < .05, Kruskal-Wallis ANOVA). When we compared groups 2 and 3 during the hearing recovery period, threshold shift differences were statistically significant at 8, 16, and 25 kHz (P < .05 for all, Mann-Whitney test) (Figure 1).
Based on the 4-grade scale previously mentioned, group 1 was graded N in all sections of all animals tested. Differences in these results were statistically significant when compared with those of group 2 or 3. When we compared groups 2 and 3 using the Mann-Whitney test, no statistical difference between them was found except at the apex, where group 3 was noted to have a more apparent loss of OHC (P = .03, Mann-Whitney test). This finding was based on a significant loss of OHC count and disarrangement of the stereocilia. At the apex, group 3 animals had grade 2 (n = 3) and grade 4 (n = 1) damage, whereas in group 2, all 4 animals did not exceed grade 1.
Light microscopy analysis of the cochlea revealed that the animals exposed to caffeine only (group 1) had preserved hair cell morphology at the organ of Corti, and the stria vascularis appeared to be intact. In the group exposed to AOSEs only (group 2), minimal changes were seen in the morphology and arrangement of the Corti tunnel, whereas the stria vascularis was preserved. In the group exposed to caffeine and AOSEs (group 3), abnormal morphology and arrangement of the Corti tunnel and the stria vascularis were present, with dilation of vessels and evidence of microscopic bleeding that might explain our results (Figure 2).
All 3 groups showed similar growth and behavior with and without daily caffeine doses of 25 mg/kg. The animals did not experience any seizures or gastroenteritis. No significant change in alertness or response to stimuli was noted. Kruskal-Wallis ANOVA did not show any difference in weight gain among the 3 groups (P > .05).
In recent years, interest has increased in studying the effects of caffeine on normal physiologic function and known medical conditions. This report addresses the effect of caffeine on ABR threshold shift in a guinea pig model exposed to AOSEs.
The study initially addressed the effect of caffeine alone on hearing. Group 1, which was exposed to caffeine for 15 days, did not show any changes on ABR threshold or on scanning electronic or light microscopy analysis. This finding is in accordance with a previously published study on caffeine by Dixit et al,14 who reported that caffeine improves the auditory pathway transmission by reducing the latency and raising the amplitudes on ABR waves.
In the pilot study,9 chronic exposure to caffeine after noise showed a change toward the delayed recovery of hearing. In that study, recovery of hearing was delayed only on 1 frequency (8 kHz) that was evident on day 14 after the noise exposure. The difference between the pilot study and the present one is that, in the pilot study, the animals were exposed to pure-tone acoustic trauma that was higher in amplitude than in the present study (120 dB compared with 110 dB). This exposure may have resulted in a permanent threshold shift in multiple frequencies, which could be one of the reasons why the difference between the groups was not more evident. Our study has demonstrated that in 3 of 4 frequencies, complete recovery of ABR threshold was noted by day 8 after the AOSE in animals not receiving caffeine.
Furthermore, the present study also used a more reasonable and clinically oriented dose of caffeine (ie, 25 mg/kg/d). In a study comparing the effect of caffeine on autoimmune encephalomyelitis in guinea pigs,10 a caffeine dose of 30 mg/kg/d was found to exert neuroprotection against the disease. In addition, in a human study,8 urinary incontinence was associated with higher doses of daily caffeine intake. In fact, the groups with extremely high caffeine intake ingested doses as high as 2.4 L per day (approximately 25-30 mg/kg/d).
The results of our study demonstrate that a daily dose of caffeine had a negative effect on hearing recovery after AOSEs at multiple frequencies. When we compared groups 2 and 3, the latter had impaired ABR threshold shifts at frequencies of 8, 16, and 25 kHz that were found to be statistically significant (P < .05 for all, Mann-Whitney test). At 20 kHz, no difference was found between both groups, likely owing to a permanent impairment at that frequency because both groups did not recover well. These results are in concordance with those of the previously published pilot study9 that showed increasing hearing impairment at 14 days after pure-tone exposure.
Another important note from this study is that the animals in groups 2 and 3 recovered in a similar manner after the first and second AOSEs. Specifically in group 2, a complete ABR threshold recovery was found in 3 of 4 frequencies. The importance of this finding is that it can be considered as the basis of animal models of other projects that address AOSEs in guinea pigs. These results are also in agreement with those of Fetoni et al,11 who noted hearing recovery from 7 to 21 days after a pure-tone–induced hearing threshold shift.
The scanning electron microscopy results showed increased OHC damage at the apex in the group that received caffeine and AOSEs (group 3) compared with the group that received AOSEs only (group 2). In addition, the Corti tunnel and stria vascularis changes on light microscopy observed in group 3 were more aggressive than the changes noted in group 2. These results are in keeping with the persistent ABR threshold shift that was noted in group 3. In the pilot study by Mujica-Mota et al,9 the scanning electronic microscopy results showed more damage to the inner hair cell. The difference in results between this study and the pilot study could be explained by differences in the caffeine dose and in the sound amplitude of AOSEs.
Many mechanisms describing how caffeine affects the auditory system in the recovery period after an AOSE have been hypothesized. The organ of Corti, lateral wall, spiral ganglion cells, and cochlear blood vessels contain high-affinity adenosine receptors.15 During an AOSE, the cochlea becomes hypoperfused and may become ischemic.16 Adenosine receptors help to promote vascular blood flow to aid the reperfusion of the cochlea.17,18 Caffeine has a nonselective adenosine receptor antagonist property, which may hinder the activities of adenosine receptors, causing further ischemia and resulting in an impaired recovery of hearing.19
Another mechanism in which caffeine may have a negative effect on hearing recovery is by increasing the release of corticosterone in response to AOSEs.20 This mechanism has been demonstrated in a human study when caffeine intake prevented the morning decline of cortisol concentration.21
The third mechanism is owing to the change in intracellular calcium levels.22 After an acoustic trauma, a significant rise in the intracellular calcium levels occurs22 that leads to apoptosis of hair cells.23 Caffeine functions by releasing additional calcium that can cause shortening of the OHCs.24,25
Our study illustrates that the additive effect of caffeine with an AOSE exposure resulted in an impaired recovery of hearing at multiple frequencies. These results are of clinical importance. Based on the results of our study, patients who continue to injest regular caffeine while being exposed to noise or after an AOSE may in fact be reducing their chances of full recovery of their hearing.
Limitations to this study include the AOSE used, which was a pure-tone sound that in real life does not represent the more common noisy environments. Another limitation was the use of a high dose of caffeine. Future studies should investigate reducing the dose of caffeine so as to identify the specific dose at which caffeine starts to affect the recovery of hearing after an AOSE. Furthermore, owing to the short follow-up (15 days), the study cannot determine the long-term effect of caffeine. Our findings could lead to further research to better understand the mechanism involving the effect of caffeine on hearing after AOSEs with the goal of lowering the general incidence of noise-induced hearing loss.
Caffeine taken in a daily dose impaired recovery of the ABR threshold shift at multiple frequencies after AOSEs in a guinea pig model. Further studies are required to determine the effects on humans and possible prophylactic strategies for noise-induced hearing loss.
Corresponding Author: Sam Joseph Daniel, MD, MSc, FRCSC, Department of Otolaryngology–Head and Neck Surgery, McGill University, Montreal, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada (email@example.com).
Accepted for Publication: December 21, 2015.
Published Online: March 3, 2016. doi:10.1001/jamaoto.2015.3938.
Author Contributions: Drs Zawawi and Daniel had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Zawawi, Mujica-Mota, Rappaport, Daniel.
Acquisition, analysis, or interpretation of data: Zawawi, Bezdjian, Daniel.
Drafting of the manuscript: Zawawi, Bezdjian, Daniel.
Critical revision of the manuscript for important intellectual content: Zawawi, Mujica-Mota, Rappaport, Daniel.
Statistical analysis: Zawawi, Daniel.
Obtained funding: Rappaport.
Administrative, technical, or material support: Zawawi, Bezdjian, Rappaport, Daniel.
Study supervision: Mujica-Mota, Rappaport, Daniel.
Conflict of Interest Disclosures: None reported.
Funding/Support: Dr Zawawi received funding support from King Abdulaziz University, Jeddah, Saudi Arabia.
Role of the Funder/Sponsor: King Abdulaziz University had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Previous Presentations: This paper was presented at the combined otolaryngology spring meeting of the American Society of Pediatric Otolaryngology; May 17, 2014; Las Vegas, Nevada.
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