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 . 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 mm ² . 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 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).

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 . 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 mm ² . 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 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).