2.1

Animals

This study was conducted on 48 adult female Sprague–Dawley rats (6 months old; weighing 250–300 g) bred at Erciyes University Experimental Clinical Research Center. The animals were housed in secure cages with unlimited access to food (pellet + tap water) at a constant temperature of 21 °C and a light–dark cycle of 12 hours in the Test Animal Laboratory of Erciyes University.

2.2

Experimental procedure

In all of the rats, the ABR thresholds were estimated after an oto-microscopic examination to evaluate normal hearing. Overall, 48 rats (96 ears) with normal hearing thresholds in the ABR estimations were included to the study. Then, 48 rats were randomized into 4 groups as follows: group 1 (n = 12) received 120 mg/kg intraperitoneal gentamicin (Genta, I.E Ulagay, Turkey); group 2 (n = 12) received 120 mg/kg intraperitoneal gentamicin plus intraperitoneal 1 ml corn oil solution (Sigma-Aldrich Chemical Co., St. Louis, MO); group 3 (n = 12) received 20 mg/kg intraperitoneal thymoquinone (Sigma-Aldrich Chemical Co., St. Louis, MO; dissolved in 1 ml corn oil solution); and group 4 (n = 12) received 120 mg/kg intraperitoneal gentamicin plus 20 mg/kg thymoquinone. All of the groups received the drugs (once daily) in the above-mentioned protocols over 15 days.

2.3

Study design

All animals were anesthetized using a combination of 100 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkey) and 7.5 mg/kg xylazine (Rompun, Bayer, Germany) via an intraperitoneal route. After anesthesia, an oto-microscopic examination was performed on 48 rats. A speculum of the appropriate size was placed into external auditory canal, followed by an examination of the external auditory canal and tympanic membranes. Debris and/or pus were removed from the external auditory canal. In the examination, no rats had external or middle ear disorders. ABR measurements were performed in both ears after oto-microscopic examination. The animals were then randomized into 4 groups and received the drugs over 15 days. No mortality or morbidity was seen after drug administration. After 15 days of using the drugs, 48 rats (96 ears) were used in ABR analyses after drug administration. The rats were sacrificed under general anesthesia. The temporal bulla of rats were bilaterally dissected and stored in 10% formaldehyde solution for histomorphological evaluation. The persons who performed ABR measurements and histopathological examination were unaware of study groups.

2.4

ABR measurement

ABR measurements were performed under general anesthesia in the ears of both rats using an interacoustic EP-25 device. The ABR responses were recorded by subdermal needle electrodes that were placed with the active electrode at vertex, the ground electrode on the glabella and reference electrodes on the right and left mastoid regions. The click stimulus was used as auditory stimuli with following settings: band-pass filters of 100–3000 Hz and a repeat rate of 21/second and also the stimulus were calibrated. The threshold was determined by starting at 70 dB and decreased by increments of 20 dB until it was approached, where 10 dB increments were instituted. Repeatability was confirmed, and the threshold determination was developed over two tests. The ABR threshold was defined on the fifth wave.

2.5

Histomorphological evaluation

The tissues that were removed were fixed in neutral formalin for 24 hours. Then, they were placed in an EDTA solution for the decalcification of osseous tissues. This was followed by an overnight washing under a water flow. The tissues were dehydrated in an alcohol series, and transparency was achieved using xylene. Serial sections (5 μm in thickness) were mounted to polylysine-coated slides. The figures were obtained from the basal turn of cochlea.

2.6

TUNEL method

An in situ apoptosis detection kit was used to detect DNA fragmentation and apoptotic cell death. Sections were stored at 60 °C overnight in an oven to facilitate deparaffinization. To complete the deparaffinization, the sections were allowed to react with xylol for 2 rounds 15 minutes each. The sections were then placed into an alcohol series of 100%, 96% and 80% by 10-minute intervals. After 2 washings in distilled water for 5 minutes, the sections were incubated with 20 μg/mL proteinase K. Next, endogen peroxidase activity was blocked in the sections, and they were allowed to react with 3% hydrogen peroxide (TA-015-HP, Lab Vision, Fremont, CA) after washing with PBS. Subsequently, the sections were incubated in balanced buffer for 15 minutes and then incubated with TdT enzyme (77 μL reaction buffer + 33 μL TdT enzyme, 1 μL TdT enzyme) at 37 °C for 60 minutes. The sections were placed in pre-warmed stopping/washing buffer at room temperature for 10 minutes before incubation with anti-digoxigenin for 45 minutes. PBS washing was performed after every step. After the washing, DAB staining was used to detect TUNEL-positive cells. For background staining, methyl green was applied for 5 minutes. The stained slides were dehydrated using an alcohol series and placed in xylol for 20 minutes. Then, the slides were covered by entellan and thin glass. Finally, the slides were evaluated using a photo-light microscope equipped with a computer.

Two observers blinded to the experimental information evaluated the TUNEL scores independently. The average number of apoptotic cells was determined by counting the TUNEL-positive cells that were in randomly chosen fields of each case. In each case, a total of one hundred both TUNEL-positive or -negative cells were counted, and the TUNEL-positive cells were given as a percentage. The cells in areas with necrosis, poor morphology or poor borders between sections were not included.

2.7

Immunohistochemical method

The remaining serial sections were assigned for immunohistochemical staining. The sections were incubated at 60 °C over night, and the slides were deparaffinized by xylene and dehydrated through alcohol series. For antigen retrieval, sections were boiled for 15 minutes in citrate buffer (10 mM, pH: 6.0) using a microwave oven. Then, they were placed in hydrogen peroxidase for 15 minutes in order to prevent endogenous peroxidase activity. The sections were incubated in blocking serum (Ultra V Block, TP-060-HL; NeoMarker, Fremont, CA) for 10 minutes and then with primary antibodies, including caspase-3 (Lab Vision) and caspase-9 (Lab Vision) in moist environment at room temperature for 60 minutes. The antigen–antibody complex was fixed using biotinylated secondary antibodies and streptavidin-peroxidase for 20 minutes. Labeling was performed using DAB, and background staining was achieved using Mayer’s hematoxylin and covered by mounting medium. The images were captured by using a camera attached to an Olympus microscope (CX31, Germany).

Control samples were processed in an identical manner, but the period of incubation with the primary antibody was omitted. Two observers blinded to the experimental information evaluated the immunolabeling scores independently. The staining intensity of the slides with their immunohistochemical protocol was graded semi-quantitatively, and the HSCORE was calculated using the following equation: HSCORE = ΣPi (i + 1), where i is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong, respectively), and where Pi is the percentage of stained cells for each intensity, varying from 0 to 100%.

2.8

Statistical analysis

SPSS for Windows 16.0 was used to analyze the findings of this study. The data were expressed as means ± SD. One way ANOVA with the Bonferroni post-hoc test were used to compare ABR results between groups before and after drug administration. Student t tests were used for the intra-group comparisons of ABR results before and after drug administration. Mann–Whitney U test was used to compare the number of TUNEL-positive cells with the caspase-3 and caspase-9 expressions among groups. p < 0.05 was considered significant.

2.1

Animals

This study was conducted on 48 adult female Sprague–Dawley rats (6 months old; weighing 250–300 g) bred at Erciyes University Experimental Clinical Research Center. The animals were housed in secure cages with unlimited access to food (pellet + tap water) at a constant temperature of 21 °C and a light–dark cycle of 12 hours in the Test Animal Laboratory of Erciyes University.

2.2

Experimental procedure

In all of the rats, the ABR thresholds were estimated after an oto-microscopic examination to evaluate normal hearing. Overall, 48 rats (96 ears) with normal hearing thresholds in the ABR estimations were included to the study. Then, 48 rats were randomized into 4 groups as follows: group 1 (n = 12) received 120 mg/kg intraperitoneal gentamicin (Genta, I.E Ulagay, Turkey); group 2 (n = 12) received 120 mg/kg intraperitoneal gentamicin plus intraperitoneal 1 ml corn oil solution (Sigma-Aldrich Chemical Co., St. Louis, MO); group 3 (n = 12) received 20 mg/kg intraperitoneal thymoquinone (Sigma-Aldrich Chemical Co., St. Louis, MO; dissolved in 1 ml corn oil solution); and group 4 (n = 12) received 120 mg/kg intraperitoneal gentamicin plus 20 mg/kg thymoquinone. All of the groups received the drugs (once daily) in the above-mentioned protocols over 15 days.

2.3

Study design

All animals were anesthetized using a combination of 100 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkey) and 7.5 mg/kg xylazine (Rompun, Bayer, Germany) via an intraperitoneal route. After anesthesia, an oto-microscopic examination was performed on 48 rats. A speculum of the appropriate size was placed into external auditory canal, followed by an examination of the external auditory canal and tympanic membranes. Debris and/or pus were removed from the external auditory canal. In the examination, no rats had external or middle ear disorders. ABR measurements were performed in both ears after oto-microscopic examination. The animals were then randomized into 4 groups and received the drugs over 15 days. No mortality or morbidity was seen after drug administration. After 15 days of using the drugs, 48 rats (96 ears) were used in ABR analyses after drug administration. The rats were sacrificed under general anesthesia. The temporal bulla of rats were bilaterally dissected and stored in 10% formaldehyde solution for histomorphological evaluation. The persons who performed ABR measurements and histopathological examination were unaware of study groups.

2.4

ABR measurement

ABR measurements were performed under general anesthesia in the ears of both rats using an interacoustic EP-25 device. The ABR responses were recorded by subdermal needle electrodes that were placed with the active electrode at vertex, the ground electrode on the glabella and reference electrodes on the right and left mastoid regions. The click stimulus was used as auditory stimuli with following settings: band-pass filters of 100–3000 Hz and a repeat rate of 21/second and also the stimulus were calibrated. The threshold was determined by starting at 70 dB and decreased by increments of 20 dB until it was approached, where 10 dB increments were instituted. Repeatability was confirmed, and the threshold determination was developed over two tests. The ABR threshold was defined on the fifth wave.

2.5

Histomorphological evaluation

The tissues that were removed were fixed in neutral formalin for 24 hours. Then, they were placed in an EDTA solution for the decalcification of osseous tissues. This was followed by an overnight washing under a water flow. The tissues were dehydrated in an alcohol series, and transparency was achieved using xylene. Serial sections (5 μm in thickness) were mounted to polylysine-coated slides. The figures were obtained from the basal turn of cochlea.

2.6

TUNEL method

An in situ apoptosis detection kit was used to detect DNA fragmentation and apoptotic cell death. Sections were stored at 60 °C overnight in an oven to facilitate deparaffinization. To complete the deparaffinization, the sections were allowed to react with xylol for 2 rounds 15 minutes each. The sections were then placed into an alcohol series of 100%, 96% and 80% by 10-minute intervals. After 2 washings in distilled water for 5 minutes, the sections were incubated with 20 μg/mL proteinase K. Next, endogen peroxidase activity was blocked in the sections, and they were allowed to react with 3% hydrogen peroxide (TA-015-HP, Lab Vision, Fremont, CA) after washing with PBS. Subsequently, the sections were incubated in balanced buffer for 15 minutes and then incubated with TdT enzyme (77 μL reaction buffer + 33 μL TdT enzyme, 1 μL TdT enzyme) at 37 °C for 60 minutes. The sections were placed in pre-warmed stopping/washing buffer at room temperature for 10 minutes before incubation with anti-digoxigenin for 45 minutes. PBS washing was performed after every step. After the washing, DAB staining was used to detect TUNEL-positive cells. For background staining, methyl green was applied for 5 minutes. The stained slides were dehydrated using an alcohol series and placed in xylol for 20 minutes. Then, the slides were covered by entellan and thin glass. Finally, the slides were evaluated using a photo-light microscope equipped with a computer.

Two observers blinded to the experimental information evaluated the TUNEL scores independently. The average number of apoptotic cells was determined by counting the TUNEL-positive cells that were in randomly chosen fields of each case. In each case, a total of one hundred both TUNEL-positive or -negative cells were counted, and the TUNEL-positive cells were given as a percentage. The cells in areas with necrosis, poor morphology or poor borders between sections were not included.

2.7

Immunohistochemical method

The remaining serial sections were assigned for immunohistochemical staining. The sections were incubated at 60 °C over night, and the slides were deparaffinized by xylene and dehydrated through alcohol series. For antigen retrieval, sections were boiled for 15 minutes in citrate buffer (10 mM, pH: 6.0) using a microwave oven. Then, they were placed in hydrogen peroxidase for 15 minutes in order to prevent endogenous peroxidase activity. The sections were incubated in blocking serum (Ultra V Block, TP-060-HL; NeoMarker, Fremont, CA) for 10 minutes and then with primary antibodies, including caspase-3 (Lab Vision) and caspase-9 (Lab Vision) in moist environment at room temperature for 60 minutes. The antigen–antibody complex was fixed using biotinylated secondary antibodies and streptavidin-peroxidase for 20 minutes. Labeling was performed using DAB, and background staining was achieved using Mayer’s hematoxylin and covered by mounting medium. The images were captured by using a camera attached to an Olympus microscope (CX31, Germany).

Control samples were processed in an identical manner, but the period of incubation with the primary antibody was omitted. Two observers blinded to the experimental information evaluated the immunolabeling scores independently. The staining intensity of the slides with their immunohistochemical protocol was graded semi-quantitatively, and the HSCORE was calculated using the following equation: HSCORE = ΣPi (i + 1), where i is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong, respectively), and where Pi is the percentage of stained cells for each intensity, varying from 0 to 100%.

2.8

Statistical analysis

SPSS for Windows 16.0 was used to analyze the findings of this study. The data were expressed as means ± SD. One way ANOVA with the Bonferroni post-hoc test were used to compare ABR results between groups before and after drug administration. Student t tests were used for the intra-group comparisons of ABR results before and after drug administration. Mann–Whitney U test was used to compare the number of TUNEL-positive cells with the caspase-3 and caspase-9 expressions among groups. p < 0.05 was considered significant.