Abstract
Objective
This study aimed to evaluate the effect of Ginkgo biloba extract (EGb 761) on noise-induced hearing loss (NIHL) by measuring audiological and biochemical parameters.
Materials and methods
Thirty-two female rats were divided into four groups (NIHL, EGb 761, NIHL + EGb 761 and control) comprising eight rats each. Groups 1 and 3 were exposed to white noise of a 100 dB sound pressure level (SPL) for 8 h, while rats in groups 2 and 3 were administered 100 mg/kg/day EGb 761 for 21 days after noise exposure. Distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) tests were performed in all groups before noise exposure and on days 1, 7, 14 and 21 after noise exposure. Intracardiac blood samples were taken on day 21 for assessment of biochemical parameters.
Results
In group 1, a significant increase was seen in audiological measurements on days 1, 7, 14 and 21 after noise exposure. In group 3, the increased DPOAE and ABR threshold decreased significantly on day 14. The total oxidant status (TOS) value was significantly higher in group 1 than in the other groups. The total antioxidant status (TAS) value was significantly higher in group 2 than in group 1. The DNA damage value was significantly higher in group 1 than in the other groups.
Conclusion
This study showed that EGb 761 is effective for treating NIHL during the early period.
1
Introduction
Noise-induced hearing loss (NIHL) is a preventable hearing problem that develops after recreational or occupational noise exposure. The disruptive effect of noise on hair cells increases cellular metabolic activity and causes cell death. The pathophysiology of NIHL involves secondary tissue damage, including both late excitotoxic tissue destruction and mechanical tissue damage. Although the molecular mechanisms of NIHL are unclear, oxidative stress factors are involved . Oxidative stress is defined as disruption of the balance between oxidant and antioxidant levels in favour of the former . Animal studies have shown that oxygen free radicals destroy hair cells following noise exposure . Although various antioxidant agents have been assessed in terms of their ability to prevent NIHL, none has been accepted as a standard treatment modality. The antioxidant EGb 761 has been used for medical purposes in China for 5000 years . EGb 761 is widely used globally as a dietary supplement to treat neurodegenerative, vascular and audiovestibular diseases. Moreover, EGb 761 can prevent myringosclerosis by scavenging free radicals . Clinical research has shown that EGb 761 can be used to treat pathologies of the inner ear and neural pathways . EGb 761 is a standardised extract produced from dried leaves of Ginkgo biloba , which contains multiple neuroprotective components effective against idiopathic sudden sensorineural hearing loss . EGb 761 has diverse pharmacological effects, such as removal of oxidative stress factors, inhibition of cyclonucleotid phosphodiesterases, membrane stabilisation, and promotion of blood flow and cognitive functions . The most important active compounds in EGb 761 are flavonoids and terpenoids. Numerous biloba extracts with standardised contents are available commercially .
The complex interactions among the various antioxidants in blood render objective measurements of their levels problematic. Nevertheless, total antioxidant status (TAS) measurements are indicative of serum antioxidant levels . Moreover, the total oxidant status (TOS) in serum can also be measured .
In this study, we evaluated the effect of EGb 761 on rats with NIHL due to increased oxidative stress. Evaluations comprised measurements of audiological (DPOAE and ABR) and biochemical (TAS, TOS, oxidative stress index (OSI), and DNA damage) parameters.
2
Materials and methods
2.1
Animals
This study was conducted after receiving approval from the Local Ethics Board for Animal Experiments. Thirty-two healthy, mature female Sprague–Dawley rats, weighing 200–240 g, were used in this study. All rats were evaluated otoscopically, and those with pathologic findings (serous otitis, acute otitis, adhesive otitis, etc. ) were excluded from the study. All rats were housed at a temperature of 21 ± 1 °C, under a 12:12 h light:dark cycle, with access to food and water ad libitum and a background noise level of < 50 dB. The rats were euthanised on day 21 after collecting blood samples for biochemical analyses.
2.2
Study groups
- Group 1:
Eight rats exposed to noise only for determination of the duration of the threshold shift.
- Group 2:
Seven rats administered EGb 761 only for evaluation of its ototoxic effects.
- Group 3:
Eight rats exposed to noise and then administered EGb 761 for evaluation of its effect on NIHL.
- Group 4:
(Control group): Eight rats not exposed to noise and not administered EGb 761.
2.3
Hearing assessment
At the beginning of the study, a pinna reflex test was performed in all rats to assess hearing. Rats were sedated by administration of ketamine (45 mg/kg) intraperitoneal [i.p.] and xylazine (5 mg/kg) i.p., and then subjected to otoscopic examination. Any obstacles that might impede the tests, such as earwax, were removed. Then, basal distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) measurements were performed on all rats. Rats with normal hearing – defined as DPOAE values 3 dB above the noise floor and ABR thresholds of 10 and 20 dB nHL – were included in the study.
2
Materials and methods
2.1
Animals
This study was conducted after receiving approval from the Local Ethics Board for Animal Experiments. Thirty-two healthy, mature female Sprague–Dawley rats, weighing 200–240 g, were used in this study. All rats were evaluated otoscopically, and those with pathologic findings (serous otitis, acute otitis, adhesive otitis, etc. ) were excluded from the study. All rats were housed at a temperature of 21 ± 1 °C, under a 12:12 h light:dark cycle, with access to food and water ad libitum and a background noise level of < 50 dB. The rats were euthanised on day 21 after collecting blood samples for biochemical analyses.
2.2
Study groups
- Group 1:
Eight rats exposed to noise only for determination of the duration of the threshold shift.
- Group 2:
Seven rats administered EGb 761 only for evaluation of its ototoxic effects.
- Group 3:
Eight rats exposed to noise and then administered EGb 761 for evaluation of its effect on NIHL.
- Group 4:
(Control group): Eight rats not exposed to noise and not administered EGb 761.
2.3
Hearing assessment
At the beginning of the study, a pinna reflex test was performed in all rats to assess hearing. Rats were sedated by administration of ketamine (45 mg/kg) intraperitoneal [i.p.] and xylazine (5 mg/kg) i.p., and then subjected to otoscopic examination. Any obstacles that might impede the tests, such as earwax, were removed. Then, basal distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) measurements were performed on all rats. Rats with normal hearing – defined as DPOAE values 3 dB above the noise floor and ABR thresholds of 10 and 20 dB nHL – were included in the study.
3
DPOAE
A GSI Audera otoacoustic emission instrument was used for DPOAE measurements. The smallest tympanometry probe was attached to the tip of the device, and measurements were carried out in a noise-insulated cabin. The measurement process was initiated after confirming that the ear probe was in a position appropriate for measurement and that the probe indicator and stimulation waveform were in the correct configuration. DPOAEs were measured using stimulations with different frequencies and intensities. Primary signal levels were adjusted to L1 = 65 dB and L2 = 55 dB for DPgram measurements. An f2/f1 ratio of 1:10 was used. DPgram measurements were carried out at frequencies of 3000, 4008, 5004, 6000, 6996, 8004, 9012, 10,008, 11,004 and 12,000 Hz. The detection threshold was defined as the primary signal level at which the DPOAE was distinguishable, at 3 dB above the noise floor. For all measurements, the responses up to the highest level of stimulation were recorded, and the test was concluded.
4
ABR
A Viasys Medelec Synergy instrument was used for ABR measurements. Measurements were performed on both ears of anaesthetised rats in a noise-insulated cabin. ABR responses were recorded using needle electrodes placed under the skin. The stimuli were delivered through insert earphones. ABR click stimuli with alternating polarities were used. The filter was adjusted to 30–1500 Hz, the repetition rate was 21.1/s and the time window was 25 ms. A total of 1024 stimuli were administered, and the signal was averaged. The threshold was defined as the lowest intensity level that could be observed and repeated. An 80 dB nHL stimulus was applied initially; the intensity was then reduced in 20 dB steps until the threshold value was reached. The magnitude of the intensity steps was reduced to 10 dB as the threshold was approached, and the threshold value was determined. At least two traces were created for each measurement, and crosschecking was performed by attempting to reproduce the threshold. The ABR threshold was defined as the lowest intensity at which wave III of ABR was observed. Baseline ABR measurements carried out prior to noise exposure were compared with those on days 1, 7, 14 and 21 after noise exposure. A single individual performed all of the audiological measurements.
4.1
Noise exposure and procedures
In previous studies, noise of different intensities, durations and characters was used to generate animal models of NIHL . In our study, rats in groups 1 and 3 were exposed to white noise at a 100 dB SPL for 8 h. DPOAE and ABR measurements were carried out in a room with a noise level of ≤ 50 dB on days 1, 7, 14 and 21 following noise exposure.
4.2
EGb 761 application
EGb 761 (100 mg/kg/day) was administered by oral gavage to the rats in groups 2 and 3 from days 1 to 21. The EGb 761 dose was selected according to Rojas et al. (2012) and is identical to that used by Konstantin Tziridis et al. (2014) in their study on the protective effects of EGb 761 against NIHL.
4.3
Biochemical parameters
On day 21, 5 mL samples of intracardiac blood were removed from the rats. Samples were added directly to heparinised tubes at 2–4 °C, and the samples were processed within 2 h to prevent DNA damage due to haemolysis. Mononuclear leukocytes were isolated by centrifugation using Histopaque 1077 (Sigma). Blood (1 mL) was placed carefully on 1 mL Histopaque and centrifuged at 500 g at 25 °C for 35 min. The mononuclear leukocyte layer was then centrifuged at 400 g for 15 min in phosphate-buffered saline (PBS). Membrane integrity was measured by trypan blue exclusion assay, and DNA damage was analysed using samples with > 90% vitality. The remaining heparinised blood was centrifuged at 1500 g for 10 min, and the serum was stored at − 80 °C for TOS and TAS analyses. TAS and TOS levels were determined using commercially available kits (Rel Assay Diagnostics; Mega Tip, Gaziantep, Turkey), and the OSI was calculated. Serum TAS and TOS levels were determined colourimetrically using a Spectramax M5 Microplate Reader (MV05047, Molecular Devices in Sunnyvale, CA) and the measurement methods developed by Erel .
TAS: In this method, Fe 2 + -o-dianisidine complex undergoes a Fenton-type reaction with hydrogen peroxide (H 2 O 2 ), resulting in formation of OH radicals. This strong reducing agent reacts with colourless o-dianisidine molecules at low temperature to form yellow-brown dianisidyl radicals. Dianisidyl radicals are involved in further oxidation reactions, increasing colour formation. However, antioxidants in the samples suppress these oxidation reactions, halting the colour formation. The result is read spectrophotometrically.
TOS: Oxidants oxidise ferrous ion-o-dianisidine complexes to ferric ions, and this reaction is accelerated ca. threefold by glycerol. Ferric ions form a colour complex with xylenol orange in the acidic medium. The intensity of the colour, which reflects the level of oxidants in the sample, is measured spectrophotometrically.
Serum TAS levels are presented in μmol Trolox equiv./L . Serum TOS assays were calibrated using H 2 O 2 , and the results are expressed as μmol H 2 O 2 equivalent/L (μmol H 2 O 2 equiv./L) . As a reliable index of oxidative stress, OSI was calculated as described previously and presented in arbitrary units.
4.4
Statistical analysis
Statistical analysis was carried out using the Statistical Package for the Social Sciences version 21.0. All quantitative variables were estimated using measures of central location ( i.e. means and medians). A one-way analysis of variance (ANOVA) was used to compare DPOAE and ABR values among the groups ( p < 0.05 was accepted as indicative of statistical significance). Differences between groups were evaluated by Bonferroni’s test. ANOVA was also used to evaluate differences in values over time among the groups ( p < 0.08 was accepted as indicative of statistical significance). The significance of differences between time points was determined using Bonferroni’s post hoc test.
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