Abstract
Purpose
This experimental study was performed to investigate the possible protective effect of pentoxifylline (PTX) on amikacin-induced ototoxicity in rats.
Materials and methods
In this study, 21 healthy female rats were randomly assigned to 1 of 3 groups: the amikacin group (n = 8), the amikacin + PTX group (n = 8), and the control group (n = 5). The amikacin group received amikacin (200 mg·kg − 1 ·day − 1 ) intramuscularly once daily for 14 days. The amikacin + PTX group received intramuscular injections of amikacin (200 mg·kg − 1 ·day − 1 ) once daily for 14 days and PTX (25 mg·kg − 1 ·day − 1 ) once daily via gastric gavage for 14 days. The control group received saline solution (1 mL·day − 1 intraperitoneal injections) once daily for 14 days. The hearing levels of the rats were evaluated using distortion product otoacoustic emissions before and after treatment.
Results
The distortion product otoacoustic emissions’ amplitude levels (decibel, sound pressure levels) measured before and after treatment at frequencies of 4000, 6000, and 8000 Hz revealed that values of the amikacin group dropped significantly at the end of treatment ( P < .01). In contrast, the amikacin + PTX and the control groups showed no significant difference at the end of the treatment compared with the initial measurements ( P > .05).
Conclusion
The results showed that PTX has protective effects on hearing functions in amikacin-induced ototoxicity in rats.
1
Introduction
Aminoglycosides are effective antibiotics with a wide spectrum that have long been used for the treatment of infections. Despite their effectiveness, aminoglycosides are associated with side effects including ototoxicity, nephrotoxicity, and vestibulotoxicity .
Amikacin, a semisynthetic first derivative of aminoglycosides , is made from the natural drug kanamycin A by acylation. Because of its structure, it is resistant to bacterial enzymes that can inactivate natural aminoglycosides such as tobramycin, kanamycin, and gentamicin . Amikacin causes the formation of free oxygen radicals leading to apoptosis. This mechanism causes ototoxicity and deafness .
Pentoxifylline (PTX), a methylxanthine derivative and phosphodiesterase inhibitor with hemorrheologic properties, affects arachidonic acid metabolism and inhibits proinflammatory mediators such as tumor necrosis factor α (TNF- α ). The TNF- α has a role in the activation of macrophages and increasing the proinflammatory secretion of neutrophils, resulting in the stimulation of apoptosis. These effects trigger cell death, resulting in necrosis of target organs. The PTX is known as an antioxidant and a free radical scavenger that decreases free oxygen radicals and nitric oxide synthase .
Amikacin is cochleotoxic, and cochlear damage can produce permanent hearing loss. Several studies have proposed various approaches for preventing aminoglycoside-induced ototoxicity, with contradictory results. In our study, we examined the possible protective effects of PTX on amikacin-induced ototoxicity.
2
Materials and methods
2.1
Animals
In this study, 21 female adult Wistar albino rats (weight, 200–240 g each) were kept in plastic cages under regular conditions of a 12:12 hours of dark-light cycle at a temperature of 21°C, with free access to food and water. All rats in this study had normal ear canals and tympanic membranes, as examined initially by otomicroscopy. Distortion product (DP) otoacoustic emissions’ (DPOAEs) recordings were performed in a quiet room. The experimental design was approved by the Istanbul University Animal Research Ethics Committee and issued approval number 15. The study was performed at the Istanbul University Experimental Medicine Institute.
2.2
Anesthesia
Ketamine hydrochloride (0.45 mg·kg − 1 ) and xylazine (5 mg·kg − 1 ) were used for anesthesia. Anesthesia was administered by intraperitoneal injections before the DPOAEs recordings.
2.3
Experimental design
Rats were assigned to 3 groups: the amikacin group (n = 8), the amikacin + PTX group (n = 8), or the control group (n = 5). The amikacin group received amikacin (200 mg·kg − 1 ·day − 1 ) intramuscularly once daily for 14 days. The amikacin + PTX group received intramuscular injections of amikacin (200 mg·kg − 1 ·day − 1 ) once daily for 14 days and PTX (25 mg·kg − 1 ·day − 1 ) once daily via gastric gavage for 14 days. The control group received saline solution (1 mL·day − 1 , intraperitoneal) once daily for 14 days. The DPOAE measurements were performed on all rats on days 0 and 15. Results taken before and after treatment were compared for each group and between groups.
2.4
The DPOAE measurements
The DPOAE measurements were performed on all rats on days 0 and 15. Otomicroscopic examination of the rats was performed before every DPOAE test to exclude middle ear pathology that might have impaired DPOAE measurements. The DPOAE measurements were taken using the smallest probe (Otodynamics Ltd, London, United Kingdom). The DPOAEs were measured as the sound pressure level using stimuli of constant intensity and frequency changes. The frequencies (f1 and f2) were adjusted to the f2:f1 ratio of 1.22 and stimulus intensity levels of 65/55 dB. Distortion product gram measurements were performed at 1001, 1501, 2002, 3003, 4004, 6006, and 7996 Hz frequencies. The DP gram was measured at 50 Hz above the frequencies of the DPOAEs. Measurements 3 dB above the frequency of 2f1 and f2 were accepted as positive. The recordings were continued until the top level was reached for every measurement. Emission values were under the noise threshold at 1001, 1501, 2002, and 3003 Hz and above it at the other frequencies. Therefore, statistical analyses were applied to the results obtained with 4000, 6000, and 8000 Hz.
2
Materials and methods
2.1
Animals
In this study, 21 female adult Wistar albino rats (weight, 200–240 g each) were kept in plastic cages under regular conditions of a 12:12 hours of dark-light cycle at a temperature of 21°C, with free access to food and water. All rats in this study had normal ear canals and tympanic membranes, as examined initially by otomicroscopy. Distortion product (DP) otoacoustic emissions’ (DPOAEs) recordings were performed in a quiet room. The experimental design was approved by the Istanbul University Animal Research Ethics Committee and issued approval number 15. The study was performed at the Istanbul University Experimental Medicine Institute.
2.2
Anesthesia
Ketamine hydrochloride (0.45 mg·kg − 1 ) and xylazine (5 mg·kg − 1 ) were used for anesthesia. Anesthesia was administered by intraperitoneal injections before the DPOAEs recordings.
2.3
Experimental design
Rats were assigned to 3 groups: the amikacin group (n = 8), the amikacin + PTX group (n = 8), or the control group (n = 5). The amikacin group received amikacin (200 mg·kg − 1 ·day − 1 ) intramuscularly once daily for 14 days. The amikacin + PTX group received intramuscular injections of amikacin (200 mg·kg − 1 ·day − 1 ) once daily for 14 days and PTX (25 mg·kg − 1 ·day − 1 ) once daily via gastric gavage for 14 days. The control group received saline solution (1 mL·day − 1 , intraperitoneal) once daily for 14 days. The DPOAE measurements were performed on all rats on days 0 and 15. Results taken before and after treatment were compared for each group and between groups.
2.4
The DPOAE measurements
The DPOAE measurements were performed on all rats on days 0 and 15. Otomicroscopic examination of the rats was performed before every DPOAE test to exclude middle ear pathology that might have impaired DPOAE measurements. The DPOAE measurements were taken using the smallest probe (Otodynamics Ltd, London, United Kingdom). The DPOAEs were measured as the sound pressure level using stimuli of constant intensity and frequency changes. The frequencies (f1 and f2) were adjusted to the f2:f1 ratio of 1.22 and stimulus intensity levels of 65/55 dB. Distortion product gram measurements were performed at 1001, 1501, 2002, 3003, 4004, 6006, and 7996 Hz frequencies. The DP gram was measured at 50 Hz above the frequencies of the DPOAEs. Measurements 3 dB above the frequency of 2f1 and f2 were accepted as positive. The recordings were continued until the top level was reached for every measurement. Emission values were under the noise threshold at 1001, 1501, 2002, and 3003 Hz and above it at the other frequencies. Therefore, statistical analyses were applied to the results obtained with 4000, 6000, and 8000 Hz.
3
Statistical analysis
Baseline and final measurements were compared for each group and among the groups. Statistical analysis of the results at 4000, 6000, and 8000 Hz were performed using the Number Cruncher Statistical System 2007 & PASS 2008 Statistical Software (Number Cruncher Statistical Systems, Kaysville, UT). Analyses included descriptive methods (mean ± SD) and 1-way analysis of variance for the intergroup analysis of normally distributed values. Tukey honestly significant difference test was used to identify group-caused differences. Baseline and final results were compared using the paired sample t test. P < .05 were considered statistically significant.
4
Results
The rats tolerated the anesthesia, amikacin, PTX, and saline administrations, respectively, and no difference was observed in the consumption of water or food among the groups.
Differences in the DPOAEs amplitudes, a measure of cochlear activity, among the 3 groups were evaluated. No statistically significant differences in DP amplitude levels were observed among the groups at the onset of the study (day 0). The mean intensities of DPs recorded on days 0 and 15 are presented in the Fig. 1 and Table 1 . The Figure 1 illustrates the amplitudes taken at 4000, 6000, and 8000 Hz before and after treatment. A comparison of the DP amplitudes before and after treatment revealed statistically significant differences in the amikacin group ( P < .01) ( Table 2 ). A comparision of the DP amplitudes measured before and after treatment revealed no statistically significant differences in the amikacin + PTX and control groups ( P > .05).
