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The effect of caffeine on hearing in a guinea pig model of acoustic trauma☆,☆☆ Mario A. Mujica-Mota, MD, MSc a , Karina Gasbarrino, BSc a , Jamie M. Rappaport, MD a, b , Robert S. Shapiro, MD a , Sam J. Daniel, MD, MSc a,⁎ a
McGill Auditory Sciences Laboratory, Montreal Children’s Hospital, Department of Otolaryngology-Head and Neck Surgery, McGill University, Montréal, Québec, Canada b Jewish General Hospital, Department of Otolaryngology-Head and Neck Surgery, Montréal, Québec, Canada
ARTI CLE I NFO
A BS TRACT
Article history:
Objective: Caffeine is a widely consumed substance affecting the metabolism of adenosine
Received 2 November 2013
and cellular metabolism of calcium. Noise also affects these metabolic pathways while inducing hearing loss. The aim of this study was to determine the effect of daily intake of caffeine on hearing loss after an episode of acoustic trauma in guinea pigs. Materials and methods: In this pilot study, forty guinea pigs were randomly divided into four groups: group I (control, n = 10) received intraperitoneal saline, group II (n = 10) received intraperitoneal caffeine (120 mg/kg/day) for 14 days, group III (n = 10) was exposed to noise (tone of 6 kHz at 120 dB for one hour) and group IV (n = 10) was exposed to noise as group III and received caffeine as group II. Auditory brainstem responses were measured at four different frequencies (8, 16, 20, and 25 kHz) prior to and at intervals of 1 h, 3 days, 10 days, and 14 days after the initial treatment. On day 14, morphological analysis was performed to assess the effects of caffeine on acoustic trauma. Results: Aggravated hearing loss was observed in group IV after 10 days of follow-up. After 14 days, one of the four frequencies (8 kHz) tested showed statistically significant greater impairment in hearing (8.2 ± 3.6 dB, p = 0.026). Auditory hair cells showed no difference while spiral ganglion cell counts were diminished in group IV (p < 0.05). Conclusion: These findings indicate that caffeine may have a detrimental effect on hearing recovery after a single event of acoustic trauma. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Given the common exposure to leisure sources of noise in young people, there is an increased concern about noiseinduced hearing loss (NIHL), which has justified increased research on the potential harm it can cause [1]. The events involved in hearing loss due to noise overexposure include: a)
mechanical trauma to the auditory hair cells and inflammation [2]; b) ischemia–reperfusion injury and c) glutamate excitotoxicity with neuronal degeneration [3]. Caffeine is commonly found in coffee and increasingly used as an additive in soda and energy drinks at extremely high concentrations. Half of the consumers of energy drinks are adolescents or young adults [4]. With a regular cup of
☆
The authors have no conflicts of interest to disclose. Disclosure. Sam J. Daniel: Merck Advisory Board. This work was supported by the McGill University Head & Neck Foundation, Sick Kids Foundation and Fonds de la Recherche en Santé du Québec. ⁎ Corresponding author at: The Montreal Children’s Hospital, 2300 Tupper Street, Rm. B-240, Montreal, QC, Canada, H3H 1P3. Tel.: + 1 514 412 4304; fax: +1 514 412 4342. E-mail address: [email protected] (S.J. Daniel). ☆☆
0196-0709/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2013.11.009
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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AM ER IC AN JOURNAL OF OTOLARYNGO LOGY –H EAD AN D N E CK ME D I CI NE AN D SUR GE RY XX ( 2 0 14 ) XXX–X XX
coffee containing between 45 and 145 mg of caffeine, the reported consumption of caffeine above 500 mg per day [5] is alarming. Furthermore, caffeine is an antagonist of adenosine [6], a nucleoside that is believed to play a protective role against cochlear cell injury [7]. Due to these facts, there is considerable interest in the potential adverse effects of caffeine on hearing. Research on this topic is highly relevant based on the fact that the consumption of caffeine-containing products is common in settings of leisure noise exposure. The objective of this pilot study was to determine whether the daily administration of caffeine can aggravate hearing loss in a guinea pig model of acoustic trauma. The working hypothesis of this study was that caffeine exacerbates the hearing loss after an episode of acoustic trauma.
2.
Materials and methods
2.1.
Animals
Forty female albino guinea pigs (weighing 300 to 400 g each) from Charles River (Wilmington, MA) were used. The animals had access to food and water ad libitum and were housed at 22 °C ± 4 °C with a 12-h light/dark cycle. The study was approved by the University Animal Care Committee. The animals were randomly assigned to four groups: group I received daily intraperitoneal (IP) injection of 4 mL saline as control (n = 10); group II received daily administration of 120 mg/ kg of IP caffeine for 14 days; group III (n = 10) was subjected to a single exposure of noise at a frequency of 6 kHz tone at 120 dB SPL for one hour. Animals from group IV (n = 10) were administered caffeine (120 mg/kg) one hour before being exposed to an identical level of noise as that given to Group III; they then continued to receive 120 mg/kg IP caffeine daily for 14 days. Distribution of animals and treatment administration were performed by a researcher who did not take part in ABR testing.
2.2.
Noise exposure
Each animal was anaesthetized with ketamine (50 mg/kg) and xylazine (1 mg/kg) and placed in a sound-proof booth. Acoustic trauma was induced by exposing the animals to a continuous frequency of 6 kHz pure tone through a generator (Intelligent Hearing Systems, Miami, FL) and amplified by an audio amplifier (D-75A, Crown Audio, Inc., Elkhart, IN). The acoustic stimulus was binaurally presented in free field by two loudspeakers (TW 034X0, Audax, France) placed 5 cm in front of the animal’s head. The sound levels were monitored by a calibrated Bruel and Kjaer Sound Level Meter Type 2230.
2.3.
Administration of caffeine
Caffeine (Sigma-Aldrich, St. Louis, MO) was injected IP, at a dose of 120 mg/kg dissolved in 4 mL of saline for 14 days. Doses were calculated daily by weight and given between 9:00 and 10:00 am. The dose chosen was determined by previous experiments in our laboratory in which doses starting from 150 mg/kg showed toxicity leading to gastroenteritis, rectal prolapse, and seizures along with enlargement of liver, as reported in previous studies [8]. Given the variable human consumption of caffeine, it was
decided to use chronic delivery at high tolerable doses. The weight and behavior of the animals were recorded every day after the initial treatment.
2.4.
Auditory brainstem response
Tests were performed under general anesthesia induced through inhalation of 5% inhaled isoflurane for induction and 2% for maintenance. Animals with abnormal ear anatomy were excluded. Auditory brainstem response (ABR) tests were performed prior to the treatment and at intervals of 1 h, 3 days, 10 days, and 14 days after the initial treatment to determine hearing threshold shifts. Bilateral testing was performed by a blinded researcher using the SmartEP System (Intelligent Hearing Systems, Miami, FL). Responses were recorded from subdermal electrodes at the tested ear (active), vertex (reference), and contralateral ear (ground). Tone burst stimuli (8, 16, 20, and 25 kHz) with Blackman envelope were presented at a rate of 39.1 bursts per second through earphones. The stimuli were presented at 80 dB SPL, decreasing in steps of 20, 10, and 5 dB to the threshold. Responses were amplified, filtered, and averaged over 1600 sweeps. Thresholds were defined as the lowest intensities from which three reproducible waves III and V were obtained. Average threshold shifts were obtained with the differences between post and the baseline test values.
2.5.
Light microscopic analysis
Immediately following the final ABR test (day 14), the cochleae were removed from every euthanized animal. Following fixation with 10% formalin for 48 h, the left cochleae were decalcified with 10% EDTA dissolved in phosphate buffered saline (0.1 M, pH 7.4) for three weeks at 4 °C. Cochleae were then dehydrated through a graded series of ethanol (50%– 100%) and embedded in paraffin for cutting into 5-μm midmodiolar sections. Slides were stained with hematoxylin and eosin and mounted for light microscopic analysis. Spiral ganglion cell (SGC) density was estimated from images of the Rosenthal’s canal at the three turns. Images were captured at 200× magnification using a Zeiss AxioCam MR3 camera (Carl Zeiss, Germany) and digitally stored using AxioVision 4.7 microscopy software. Images at the basal, middle, and apical turns were processed with NIH ImageJ program (US National Institutes of Health). The bony boundaries of the Rosenthal canal were marked. The cell bodies of spiral ganglion neurons were manually counted and density was calculated by dividing the number of neurons per section by the cross-sectional area of the Rosenthal’s canal in each section and expressed as the number of cells per mm2. The SGC densities were averaged to obtain final values of each turn.
2.6.
Scanning electron microscopic analysis
The right cochleae were fixed in 2.5% glutaraldehyde for 2 h. The tissue was then soaked in 0.1 M PBS solution for 24 h at 4 °C. The cochleae were post-fixated in osmium tetroxide for 90 min and dehydrated in graded solutions up to 70% ethanol. The cochleae were drilled, the organ of Corti dissected and the bone covering removed. The samples were further dehydrated
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
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in solutions up to 100% ethanol. Then the samples were critical point dried, mounted, and sputter coated with gold. A field emission scanning electron microscope was used for qualitative analysis (Hitachi S4700; Hitachi, Japan).
2.7.
Statistical analysis
The mean difference between each measurement and the baseline for each frequency (8, 16, 20, and 25 kHz) was calculated. Threshold shifts were analyzed with repeated measures ANOVA in which treatment was a between-subjects variable. A one-way ANOVA was used to compare the weights and SGC densities between groups. Tukey’s HSD test was used to compare statistical significance between two groups in the analyses. Statistical significance was set at P ≤ 0.05. Analyses were performed using JMP 10 (SAS Inc. Cary, NC).
3.
Results
3.1.
Threshold shifts
Repeated measures ANOVA showed a significant difference between groups (F = 67.63, p < 0.0001; and group-frequency interaction, F = 13.16, p < 0.0001). Analysis of the baseline thresholds between the four groups (Fig. 1) showed no significant differences at the four frequencies. The hearing thresholds in the caffeine group (group II) remained unchanged from baseline to the end of the follow-up period. At 1 h and 3 days following exposure to noise, there were no statistically significant differences in threshold shift between the noise group (group III) and caffeine plus noise group (group IV). Despite a progressive recovery throughout the 14 days, post-hoc analysis at day 10 showed that group IV had greater thresholds shifts than group III at 8 kHz (11.5 ± 3.6 dB, CI 4.2– 18.7, p = 0.002), 16 kHz (13 ± 3.45 dB, CI 0.88–25.1, p = 0.022) and 20 kHz (13.15 ± 2.82 dB, CI 3.22–23.07, p = 0.001). After 14 days, the difference in threshold shifts between these two groups was still persistent and significant at 8 kHz (8.2 ± 3.6 dB, CI 0.98–15.5, p = 0.026).
Fig. 1 – ABR threshold shifts at baseline, 1 h, 3 days, 10 days, and 14 days of the treatment. dB, decibels; kHz, kilohertz. * Significant differences, p < 0.05. Bars represent mean ± SEM.
Fig. 2 – Weight gain differences between groups. Significant differences were found between Caffeine plus Noise group versus Control (p = 0.0097) and Caffeine alone (p = 0.0019).
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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statistically significant differences between caffeine plus noise versus control group (p = 0.0097) and between caffeine plus noise versus caffeine alone group (p = 0.0019). Animals subjected solely to either caffeine or noise had decreased but not statistically significant different weight gain when compared with the control group. Animals subjected to noise plus caffeine had no overall weight gain during the evaluation period (Fig. 2).
3.3. Histological analysis and Scanning Electron Microscopy Caffeine alone did not have an effect on SGC density as seen in results of group II. A decrease in SGC counts was noticed in group IV, having statistically significant differences with group III at the middle (p = 0.014) and apical turns (p = 0.046) (Fig. 3). Scanning electron microscopy did not reveal significant differences between the four groups. However, groups subjected to noise showed disarrangement of hair-cells’ stereocilia predominantly in the row of inner hair cells (Fig. 4).
4.
Fig. 3 – Spiral ganglion neuron counts. Top panel: Counts at the three turns of the cochleae. * p < 0.05. Bars represent mean ± SEM. Bottom panel: Spiral ganglion cells. H–E staining. Control (A), Caffeine (B), Noise (C), and Caffeine plus Noise (D).
3.2.
Animal behavior and body weights
The animals, administered with caffeine at doses of 120 mg/ kg in both the groups (II and IV), were more responsive to stimuli in terms of alertness and excitability, which is a normal physiological response to caffeine. None of the animals developed gastroenteritis, or seizures and there was no liver enlargement found on autopsies of the animals subjected to higher doses. In terms of body weight, one-way ANOVA revealed statistically significant differences between the groups (p = 0.0019). Post-hoc analysis revealed only
Discussion
This study reports the chronological changes in ABR thresholds following exposure to high intensity noise and administration of caffeine. Similar to the results of another study infusing caffeine in the perilymph compartment [9], this study did not find caffeine to have a deleterious effect on hearing even after 14 days of intraperitoneal administration of caffeine. A human study showed that caffeine can improve transmission in the auditory pathways by decreasing the latency and increasing the amplitude of waves in the ABR [10]. However, this study used a single low dose of caffeine (3 mg/kg), disregarding the effects of repetitive consumption of this compound. In the current study, which examines chronic exposure to caffeine, there was a tendency towards higher hearing thresholds in the group treated with caffeine plus noise when compared with the noise only group. The results showed that the caffeine plus noise group suffered significantly greater hearing loss than the noise alone group at 16 kHz and 20 kHz after 10 days. At day 14, significant differences were still observed at one particular frequency (8 kHz). As suggested by other studies, after an episode of acoustic trauma follows an early phase of free radical production, which continues until it peaks at day 7–10 post-exposure [11]. The observation that the differences were significant after 10 days suggests that caffeine might have a detrimental effect in this phase. Regarding the frequencies involved, as reported in previous studies using the same model, the hearing frequencies ranging from 6 to 20 kHz were primarily affected in the early stress period [12]. With a prolonged follow-up time (7 to 21 days), these frequencies show a recovery from noise with a permanent threshold shift of 15 to 30 dB with predominance between the 6 and 12 kHz frequency range [13]. In addition, studies have shown that after acoustic trauma from a pure tone noise exposure, subjects present two maximum threshold
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
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Fig. 4 – Scanning Electron Microscopy images. Control (A), Caffeine (B), Noise (C), and Caffeine plus Noise (D).
shifts peaks at: a) one octave above the pure tone frequency, and b) another one beyond with different frequency positions among different animal species [14]. There are four main mechanisms that might be responsible for the effects of caffeine on noise-induced hearing loss observed in these experiments. One possibility is through a rise in level of intracellular calcium, a mechanism considered to contribute to cochlear injury after noise exposure [15]. Calcium promotes the activation of phospholipase A2 calcium-dependant isoforms and the calpain-dependent cleavage of calcineurin, which ultimately lead to apoptosis [16]. This theory has been supported by studies attenuating NIHL with calcium channel blockers and lowering the concentration of calcium in perilymph [17,18]. Moreover, studies have shown that caffeine can cause shortening of outer hair cells [19] by releasing calcium from intracellular stores secondary to the activation of ryanodine receptors [20]. Furthermore, these effects have been observed in supporting cells of the organ of Corti, affecting hair cell motility [21]. Based on these studies, it might be possible that caffeine enhances the rise of intracellular calcium already initiated by noise. A second mechanism possibly involved is the nonselective adenosine receptor antagonist properties of caffeine [22]. Adenosine is an endogenous purine nucleoside whose levels increase after cellular stresses in the nervous system such as ischemia or excitotoxicity [23]. The expression of adenosine receptors in the cochlea has been previously characterized [24], suggesting that high-affinity adenosine receptors (A1, A2A, A3) are present in the organ of Corti, SGC, lateral wall, and cochlear blood vessels [25]. The neuroprotective effects of adenosine are believed to be mediated by the activation of A1 receptors that are linked to G-proteins, which
inhibit adenylate cyclase thereby impeding the release of glutamate, an excitotoxic neurotransmitter [23]. Previous studies have reported acute SGC degeneration after noise exposure [3,26]. This is consistent with our findings of lower SGC density in the group that received caffeine plus noise, where excitotoxic damage may have been exacerbated by caffeine. In support of this hypothesis, animal studies have shown that adenosine receptor agonists can protect the cochlea against cisplatin ototoxicity [27] and transient ischemia [28] through the blockage of production of reactive oxygen species and redistribution of oxidative enzymes in the subcellular compartment [29]. Furthermore, acoustic trauma has been reported to induce up-regulation of A1 receptors in the organ of Corti [30], which might serve to increase the efficacy of adenosine after cell stress. Previous studies have shown that A1 agonists mitigated ABR threshold shifts and reduced oxidative damage in the cochlea after acoustic trauma [31–33]. Therefore, in the current experiments, caffeine might have antagonized these receptors. Based on the observations in histological analysis, this mechanism might better explain the damage caused to spiral ganglion cells than to the auditory hair cells. A third possible mechanism possibly involved is the noiseinduced hypoperfusion and ischemia in the cochlea [34]. As reported by other researchers, noise exposure can enhance cochlear metabolic activity, with increased consumption of ATP and adenosine production [35]. Adenosine can also stimulate cochlear blood flow [36] through the A2A receptors that are found in high concentrations in vascular tissue [37]. Supporting this hypothesis, a number of studies have reported that caffeine can promote a reduction in cerebral blood flow and arteriole diameter in humans [38], an effect that might be extensive in the spiral modiolar arteries as
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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observed in vitro studies [39]. Thus, blockage of these receptors by caffeine could have exacerbated the hypoxic environment of NIHL [40]. A fourth possible mechanism is the exacerbation, by caffeine, of the physiological increase in corticosterone, as an acute response to noise [41]. Studies in human subjects have demonstrated that caffeine intake at doses of 3 mg/kg of body weight prevented the fall of morning concentrations of cortisol [42] through the alteration of the hypothalamic–pituitary–adrenocortical axis (HPAA) as seen in animal models subjected to subacute administration of caffeine [43]. A study assessing the activation of the HPAA in rats by subjecting them to noise (80 db and 105 dB) followed by a single subcutaneous injection of caffeine, showed that the levels of cortisol were elevated when both the stimuli were given separately [44]. However, when given in combination, caffeine did not significantly affect the levels of stress hormones following noise exposure. The authors suggested that these results could be due to a ceiling effect of the hormonal responses. Given this evidence, the effects of HPAA alteration due to noise during prolonged exposures to caffeine or noise alone and its effects on hearing are still unknown. Our findings suggest that caffeine alone does not have a deleterious effect on hearing, which is similar to the reported effects of adenosine agonists alone [45]. Our results showed that the animals that were given caffeine and noise in combination had greater hearing thresholds than those that received noise alone. These animals exhibited decreased SGC density while hair cell morphology was preserved intact. These results could only be explained by a combination of the mechanisms previously detailed. Despite a relatively small difference between these groups (8 dB SPL), this can have greater implications when noise exposure is repetitive. Our results suggest that an individual could be reducing his/her potential recovery from acoustic trauma by having a high intake of caffeine. Nevertheless, limitations that make our pilot study difficult to extrapolate to the human counterparts are the short follow-up (which might preclude observing full recovery) and the large caffeine dose used. Further studies are being planned to investigate the questions that arose in this pilot study.
5.
Conclusions
Caffeine attenuated/impaired the recovery of hearing after 14 days following an event of acoustic trauma. Spiral ganglion cell density was lower in the group treated with caffeine and noise in the apex and middle turns of the cochlea while hair cell morphology remained intact. Further research is required to determine the long term effects of caffeine on noiseinduced hearing loss.
Acknowledgments The authors wish to thank Dr. Anna Rita Fetoni from the Institute of Otolaryngology at the Catholic University in Rome for her assistance with the set-up of the experiments.
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Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
The effect of caffeine on hearing in a guinea pig model of acoustic trauma☆,☆☆ Mario A. Mujica-Mota, MD, MSc a , Karina Gasbarrino, BSc a , Jamie M. Rappaport, MD a, b , Robert S. Shapiro, MD a , Sam J. Daniel, MD, MSc a,⁎ a
McGill Auditory Sciences Laboratory, Montreal Children’s Hospital, Department of Otolaryngology-Head and Neck Surgery, McGill University, Montréal, Québec, Canada b Jewish General Hospital, Department of Otolaryngology-Head and Neck Surgery, Montréal, Québec, Canada
ARTI CLE I NFO
A BS TRACT
Article history:
Objective: Caffeine is a widely consumed substance affecting the metabolism of adenosine
Received 2 November 2013
and cellular metabolism of calcium. Noise also affects these metabolic pathways while inducing hearing loss. The aim of this study was to determine the effect of daily intake of caffeine on hearing loss after an episode of acoustic trauma in guinea pigs. Materials and methods: In this pilot study, forty guinea pigs were randomly divided into four groups: group I (control, n = 10) received intraperitoneal saline, group II (n = 10) received intraperitoneal caffeine (120 mg/kg/day) for 14 days, group III (n = 10) was exposed to noise (tone of 6 kHz at 120 dB for one hour) and group IV (n = 10) was exposed to noise as group III and received caffeine as group II. Auditory brainstem responses were measured at four different frequencies (8, 16, 20, and 25 kHz) prior to and at intervals of 1 h, 3 days, 10 days, and 14 days after the initial treatment. On day 14, morphological analysis was performed to assess the effects of caffeine on acoustic trauma. Results: Aggravated hearing loss was observed in group IV after 10 days of follow-up. After 14 days, one of the four frequencies (8 kHz) tested showed statistically significant greater impairment in hearing (8.2 ± 3.6 dB, p = 0.026). Auditory hair cells showed no difference while spiral ganglion cell counts were diminished in group IV (p < 0.05). Conclusion: These findings indicate that caffeine may have a detrimental effect on hearing recovery after a single event of acoustic trauma. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Given the common exposure to leisure sources of noise in young people, there is an increased concern about noiseinduced hearing loss (NIHL), which has justified increased research on the potential harm it can cause [1]. The events involved in hearing loss due to noise overexposure include: a)
mechanical trauma to the auditory hair cells and inflammation [2]; b) ischemia–reperfusion injury and c) glutamate excitotoxicity with neuronal degeneration [3]. Caffeine is commonly found in coffee and increasingly used as an additive in soda and energy drinks at extremely high concentrations. Half of the consumers of energy drinks are adolescents or young adults [4]. With a regular cup of
☆
The authors have no conflicts of interest to disclose. Disclosure. Sam J. Daniel: Merck Advisory Board. This work was supported by the McGill University Head & Neck Foundation, Sick Kids Foundation and Fonds de la Recherche en Santé du Québec. ⁎ Corresponding author at: The Montreal Children’s Hospital, 2300 Tupper Street, Rm. B-240, Montreal, QC, Canada, H3H 1P3. Tel.: + 1 514 412 4304; fax: +1 514 412 4342. E-mail address: [email protected] (S.J. Daniel). ☆☆
0196-0709/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2013.11.009
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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coffee containing between 45 and 145 mg of caffeine, the reported consumption of caffeine above 500 mg per day [5] is alarming. Furthermore, caffeine is an antagonist of adenosine [6], a nucleoside that is believed to play a protective role against cochlear cell injury [7]. Due to these facts, there is considerable interest in the potential adverse effects of caffeine on hearing. Research on this topic is highly relevant based on the fact that the consumption of caffeine-containing products is common in settings of leisure noise exposure. The objective of this pilot study was to determine whether the daily administration of caffeine can aggravate hearing loss in a guinea pig model of acoustic trauma. The working hypothesis of this study was that caffeine exacerbates the hearing loss after an episode of acoustic trauma.
2.
Materials and methods
2.1.
Animals
Forty female albino guinea pigs (weighing 300 to 400 g each) from Charles River (Wilmington, MA) were used. The animals had access to food and water ad libitum and were housed at 22 °C ± 4 °C with a 12-h light/dark cycle. The study was approved by the University Animal Care Committee. The animals were randomly assigned to four groups: group I received daily intraperitoneal (IP) injection of 4 mL saline as control (n = 10); group II received daily administration of 120 mg/ kg of IP caffeine for 14 days; group III (n = 10) was subjected to a single exposure of noise at a frequency of 6 kHz tone at 120 dB SPL for one hour. Animals from group IV (n = 10) were administered caffeine (120 mg/kg) one hour before being exposed to an identical level of noise as that given to Group III; they then continued to receive 120 mg/kg IP caffeine daily for 14 days. Distribution of animals and treatment administration were performed by a researcher who did not take part in ABR testing.
2.2.
Noise exposure
Each animal was anaesthetized with ketamine (50 mg/kg) and xylazine (1 mg/kg) and placed in a sound-proof booth. Acoustic trauma was induced by exposing the animals to a continuous frequency of 6 kHz pure tone through a generator (Intelligent Hearing Systems, Miami, FL) and amplified by an audio amplifier (D-75A, Crown Audio, Inc., Elkhart, IN). The acoustic stimulus was binaurally presented in free field by two loudspeakers (TW 034X0, Audax, France) placed 5 cm in front of the animal’s head. The sound levels were monitored by a calibrated Bruel and Kjaer Sound Level Meter Type 2230.
2.3.
Administration of caffeine
Caffeine (Sigma-Aldrich, St. Louis, MO) was injected IP, at a dose of 120 mg/kg dissolved in 4 mL of saline for 14 days. Doses were calculated daily by weight and given between 9:00 and 10:00 am. The dose chosen was determined by previous experiments in our laboratory in which doses starting from 150 mg/kg showed toxicity leading to gastroenteritis, rectal prolapse, and seizures along with enlargement of liver, as reported in previous studies [8]. Given the variable human consumption of caffeine, it was
decided to use chronic delivery at high tolerable doses. The weight and behavior of the animals were recorded every day after the initial treatment.
2.4.
Auditory brainstem response
Tests were performed under general anesthesia induced through inhalation of 5% inhaled isoflurane for induction and 2% for maintenance. Animals with abnormal ear anatomy were excluded. Auditory brainstem response (ABR) tests were performed prior to the treatment and at intervals of 1 h, 3 days, 10 days, and 14 days after the initial treatment to determine hearing threshold shifts. Bilateral testing was performed by a blinded researcher using the SmartEP System (Intelligent Hearing Systems, Miami, FL). Responses were recorded from subdermal electrodes at the tested ear (active), vertex (reference), and contralateral ear (ground). Tone burst stimuli (8, 16, 20, and 25 kHz) with Blackman envelope were presented at a rate of 39.1 bursts per second through earphones. The stimuli were presented at 80 dB SPL, decreasing in steps of 20, 10, and 5 dB to the threshold. Responses were amplified, filtered, and averaged over 1600 sweeps. Thresholds were defined as the lowest intensities from which three reproducible waves III and V were obtained. Average threshold shifts were obtained with the differences between post and the baseline test values.
2.5.
Light microscopic analysis
Immediately following the final ABR test (day 14), the cochleae were removed from every euthanized animal. Following fixation with 10% formalin for 48 h, the left cochleae were decalcified with 10% EDTA dissolved in phosphate buffered saline (0.1 M, pH 7.4) for three weeks at 4 °C. Cochleae were then dehydrated through a graded series of ethanol (50%– 100%) and embedded in paraffin for cutting into 5-μm midmodiolar sections. Slides were stained with hematoxylin and eosin and mounted for light microscopic analysis. Spiral ganglion cell (SGC) density was estimated from images of the Rosenthal’s canal at the three turns. Images were captured at 200× magnification using a Zeiss AxioCam MR3 camera (Carl Zeiss, Germany) and digitally stored using AxioVision 4.7 microscopy software. Images at the basal, middle, and apical turns were processed with NIH ImageJ program (US National Institutes of Health). The bony boundaries of the Rosenthal canal were marked. The cell bodies of spiral ganglion neurons were manually counted and density was calculated by dividing the number of neurons per section by the cross-sectional area of the Rosenthal’s canal in each section and expressed as the number of cells per mm2. The SGC densities were averaged to obtain final values of each turn.
2.6.
Scanning electron microscopic analysis
The right cochleae were fixed in 2.5% glutaraldehyde for 2 h. The tissue was then soaked in 0.1 M PBS solution for 24 h at 4 °C. The cochleae were post-fixated in osmium tetroxide for 90 min and dehydrated in graded solutions up to 70% ethanol. The cochleae were drilled, the organ of Corti dissected and the bone covering removed. The samples were further dehydrated
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
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in solutions up to 100% ethanol. Then the samples were critical point dried, mounted, and sputter coated with gold. A field emission scanning electron microscope was used for qualitative analysis (Hitachi S4700; Hitachi, Japan).
2.7.
Statistical analysis
The mean difference between each measurement and the baseline for each frequency (8, 16, 20, and 25 kHz) was calculated. Threshold shifts were analyzed with repeated measures ANOVA in which treatment was a between-subjects variable. A one-way ANOVA was used to compare the weights and SGC densities between groups. Tukey’s HSD test was used to compare statistical significance between two groups in the analyses. Statistical significance was set at P ≤ 0.05. Analyses were performed using JMP 10 (SAS Inc. Cary, NC).
3.
Results
3.1.
Threshold shifts
Repeated measures ANOVA showed a significant difference between groups (F = 67.63, p < 0.0001; and group-frequency interaction, F = 13.16, p < 0.0001). Analysis of the baseline thresholds between the four groups (Fig. 1) showed no significant differences at the four frequencies. The hearing thresholds in the caffeine group (group II) remained unchanged from baseline to the end of the follow-up period. At 1 h and 3 days following exposure to noise, there were no statistically significant differences in threshold shift between the noise group (group III) and caffeine plus noise group (group IV). Despite a progressive recovery throughout the 14 days, post-hoc analysis at day 10 showed that group IV had greater thresholds shifts than group III at 8 kHz (11.5 ± 3.6 dB, CI 4.2– 18.7, p = 0.002), 16 kHz (13 ± 3.45 dB, CI 0.88–25.1, p = 0.022) and 20 kHz (13.15 ± 2.82 dB, CI 3.22–23.07, p = 0.001). After 14 days, the difference in threshold shifts between these two groups was still persistent and significant at 8 kHz (8.2 ± 3.6 dB, CI 0.98–15.5, p = 0.026).
Fig. 1 – ABR threshold shifts at baseline, 1 h, 3 days, 10 days, and 14 days of the treatment. dB, decibels; kHz, kilohertz. * Significant differences, p < 0.05. Bars represent mean ± SEM.
Fig. 2 – Weight gain differences between groups. Significant differences were found between Caffeine plus Noise group versus Control (p = 0.0097) and Caffeine alone (p = 0.0019).
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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statistically significant differences between caffeine plus noise versus control group (p = 0.0097) and between caffeine plus noise versus caffeine alone group (p = 0.0019). Animals subjected solely to either caffeine or noise had decreased but not statistically significant different weight gain when compared with the control group. Animals subjected to noise plus caffeine had no overall weight gain during the evaluation period (Fig. 2).
3.3. Histological analysis and Scanning Electron Microscopy Caffeine alone did not have an effect on SGC density as seen in results of group II. A decrease in SGC counts was noticed in group IV, having statistically significant differences with group III at the middle (p = 0.014) and apical turns (p = 0.046) (Fig. 3). Scanning electron microscopy did not reveal significant differences between the four groups. However, groups subjected to noise showed disarrangement of hair-cells’ stereocilia predominantly in the row of inner hair cells (Fig. 4).
4.
Fig. 3 – Spiral ganglion neuron counts. Top panel: Counts at the three turns of the cochleae. * p < 0.05. Bars represent mean ± SEM. Bottom panel: Spiral ganglion cells. H–E staining. Control (A), Caffeine (B), Noise (C), and Caffeine plus Noise (D).
3.2.
Animal behavior and body weights
The animals, administered with caffeine at doses of 120 mg/ kg in both the groups (II and IV), were more responsive to stimuli in terms of alertness and excitability, which is a normal physiological response to caffeine. None of the animals developed gastroenteritis, or seizures and there was no liver enlargement found on autopsies of the animals subjected to higher doses. In terms of body weight, one-way ANOVA revealed statistically significant differences between the groups (p = 0.0019). Post-hoc analysis revealed only
Discussion
This study reports the chronological changes in ABR thresholds following exposure to high intensity noise and administration of caffeine. Similar to the results of another study infusing caffeine in the perilymph compartment [9], this study did not find caffeine to have a deleterious effect on hearing even after 14 days of intraperitoneal administration of caffeine. A human study showed that caffeine can improve transmission in the auditory pathways by decreasing the latency and increasing the amplitude of waves in the ABR [10]. However, this study used a single low dose of caffeine (3 mg/kg), disregarding the effects of repetitive consumption of this compound. In the current study, which examines chronic exposure to caffeine, there was a tendency towards higher hearing thresholds in the group treated with caffeine plus noise when compared with the noise only group. The results showed that the caffeine plus noise group suffered significantly greater hearing loss than the noise alone group at 16 kHz and 20 kHz after 10 days. At day 14, significant differences were still observed at one particular frequency (8 kHz). As suggested by other studies, after an episode of acoustic trauma follows an early phase of free radical production, which continues until it peaks at day 7–10 post-exposure [11]. The observation that the differences were significant after 10 days suggests that caffeine might have a detrimental effect in this phase. Regarding the frequencies involved, as reported in previous studies using the same model, the hearing frequencies ranging from 6 to 20 kHz were primarily affected in the early stress period [12]. With a prolonged follow-up time (7 to 21 days), these frequencies show a recovery from noise with a permanent threshold shift of 15 to 30 dB with predominance between the 6 and 12 kHz frequency range [13]. In addition, studies have shown that after acoustic trauma from a pure tone noise exposure, subjects present two maximum threshold
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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Fig. 4 – Scanning Electron Microscopy images. Control (A), Caffeine (B), Noise (C), and Caffeine plus Noise (D).
shifts peaks at: a) one octave above the pure tone frequency, and b) another one beyond with different frequency positions among different animal species [14]. There are four main mechanisms that might be responsible for the effects of caffeine on noise-induced hearing loss observed in these experiments. One possibility is through a rise in level of intracellular calcium, a mechanism considered to contribute to cochlear injury after noise exposure [15]. Calcium promotes the activation of phospholipase A2 calcium-dependant isoforms and the calpain-dependent cleavage of calcineurin, which ultimately lead to apoptosis [16]. This theory has been supported by studies attenuating NIHL with calcium channel blockers and lowering the concentration of calcium in perilymph [17,18]. Moreover, studies have shown that caffeine can cause shortening of outer hair cells [19] by releasing calcium from intracellular stores secondary to the activation of ryanodine receptors [20]. Furthermore, these effects have been observed in supporting cells of the organ of Corti, affecting hair cell motility [21]. Based on these studies, it might be possible that caffeine enhances the rise of intracellular calcium already initiated by noise. A second mechanism possibly involved is the nonselective adenosine receptor antagonist properties of caffeine [22]. Adenosine is an endogenous purine nucleoside whose levels increase after cellular stresses in the nervous system such as ischemia or excitotoxicity [23]. The expression of adenosine receptors in the cochlea has been previously characterized [24], suggesting that high-affinity adenosine receptors (A1, A2A, A3) are present in the organ of Corti, SGC, lateral wall, and cochlear blood vessels [25]. The neuroprotective effects of adenosine are believed to be mediated by the activation of A1 receptors that are linked to G-proteins, which
inhibit adenylate cyclase thereby impeding the release of glutamate, an excitotoxic neurotransmitter [23]. Previous studies have reported acute SGC degeneration after noise exposure [3,26]. This is consistent with our findings of lower SGC density in the group that received caffeine plus noise, where excitotoxic damage may have been exacerbated by caffeine. In support of this hypothesis, animal studies have shown that adenosine receptor agonists can protect the cochlea against cisplatin ototoxicity [27] and transient ischemia [28] through the blockage of production of reactive oxygen species and redistribution of oxidative enzymes in the subcellular compartment [29]. Furthermore, acoustic trauma has been reported to induce up-regulation of A1 receptors in the organ of Corti [30], which might serve to increase the efficacy of adenosine after cell stress. Previous studies have shown that A1 agonists mitigated ABR threshold shifts and reduced oxidative damage in the cochlea after acoustic trauma [31–33]. Therefore, in the current experiments, caffeine might have antagonized these receptors. Based on the observations in histological analysis, this mechanism might better explain the damage caused to spiral ganglion cells than to the auditory hair cells. A third possible mechanism possibly involved is the noiseinduced hypoperfusion and ischemia in the cochlea [34]. As reported by other researchers, noise exposure can enhance cochlear metabolic activity, with increased consumption of ATP and adenosine production [35]. Adenosine can also stimulate cochlear blood flow [36] through the A2A receptors that are found in high concentrations in vascular tissue [37]. Supporting this hypothesis, a number of studies have reported that caffeine can promote a reduction in cerebral blood flow and arteriole diameter in humans [38], an effect that might be extensive in the spiral modiolar arteries as
Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
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observed in vitro studies [39]. Thus, blockage of these receptors by caffeine could have exacerbated the hypoxic environment of NIHL [40]. A fourth possible mechanism is the exacerbation, by caffeine, of the physiological increase in corticosterone, as an acute response to noise [41]. Studies in human subjects have demonstrated that caffeine intake at doses of 3 mg/kg of body weight prevented the fall of morning concentrations of cortisol [42] through the alteration of the hypothalamic–pituitary–adrenocortical axis (HPAA) as seen in animal models subjected to subacute administration of caffeine [43]. A study assessing the activation of the HPAA in rats by subjecting them to noise (80 db and 105 dB) followed by a single subcutaneous injection of caffeine, showed that the levels of cortisol were elevated when both the stimuli were given separately [44]. However, when given in combination, caffeine did not significantly affect the levels of stress hormones following noise exposure. The authors suggested that these results could be due to a ceiling effect of the hormonal responses. Given this evidence, the effects of HPAA alteration due to noise during prolonged exposures to caffeine or noise alone and its effects on hearing are still unknown. Our findings suggest that caffeine alone does not have a deleterious effect on hearing, which is similar to the reported effects of adenosine agonists alone [45]. Our results showed that the animals that were given caffeine and noise in combination had greater hearing thresholds than those that received noise alone. These animals exhibited decreased SGC density while hair cell morphology was preserved intact. These results could only be explained by a combination of the mechanisms previously detailed. Despite a relatively small difference between these groups (8 dB SPL), this can have greater implications when noise exposure is repetitive. Our results suggest that an individual could be reducing his/her potential recovery from acoustic trauma by having a high intake of caffeine. Nevertheless, limitations that make our pilot study difficult to extrapolate to the human counterparts are the short follow-up (which might preclude observing full recovery) and the large caffeine dose used. Further studies are being planned to investigate the questions that arose in this pilot study.
5.
Conclusions
Caffeine attenuated/impaired the recovery of hearing after 14 days following an event of acoustic trauma. Spiral ganglion cell density was lower in the group treated with caffeine and noise in the apex and middle turns of the cochlea while hair cell morphology remained intact. Further research is required to determine the long term effects of caffeine on noiseinduced hearing loss.
Acknowledgments The authors wish to thank Dr. Anna Rita Fetoni from the Institute of Otolaryngology at the Catholic University in Rome for her assistance with the set-up of the experiments.
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Please cite this article as: Mujica-Mota MA, et al, The effect of caffeine on hearing in a guinea pig model of acoustic trauma, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2013.11.009
