Abstract
Objective
The aim of this experimental study was to investigate the efficacy of caffeic acid phenethyl ester (CAPE) in the prevention of streptomycin-induced ototoxicity.
Materials and Methods
Thirty-two adult Wistar albino rats were divided into 4 groups: control (n = 8), streptomycin (n = 8), CAPE (n = 8), and streptomycin + CAPE (n = 8). Rats were tested with distortion product otoacoustic emissions (DPOAEs) before drug administration. The animals in all groups were killed under general anesthesia on the 45th day following last DPOAE measurements. Hearing results were analyzed statistically to determine differences in amplitudes of DPOAE. Also, the cochleas of each rat were evaluated by histopathological and immunohistochemical examination.
Results
Significant difference was not observed in cochlear hair cells in the control and CAPE groups. In the streptomycin group, severe degeneration of hair cells and increased apoptotic cells were observed. In the streptomycin + CAPE group, although some deteriorations were observed, hair cells were mostly preserved. The DPgram of the streptomycin and streptomycin + CAPE groups was significantly deteriorated ( P < .05). The analysis of the DPgram results revealed statistically significant differences between the groups of streptomycin and streptomycin + CAPE ( P < .05).
Conclusions
Caffeic acid phenethyl ester treatment attenuated hair cells injury in the inner ear, possibly via its antioxidant effect. Prophylactic administration of CAPE for streptomycin ototoxicity ameliorated hearing deterioration in rats.
1
Introduction
Class of aminoglycoside antibiotic is the first and the most common agent implicated as having ototoxic adverse effects . Although the true incidence is difficult to determine, the reported incidence of significant ototoxicity from aminoglycosides varies widely in the literature and ranges from 2% to 5% . Because of well-known cochleotoxic or vestibulotoxic adverse effects, in recent decades, especially in industrialized countries, the use of aminoglycosides has been relatively limited, several are only used topically, and many aminoglycoside drugs are no longer in use . In developed countries, class of aminoglycoside antibiotic currently tends to be reserved for clinical use only in cases of resistant tuberculosis because of the need for life-saving treatment . On the other hand, aminoglycosides are one of the most commonly prescribed antibiotics in other parts of the world, mostly in developing countries and underdeveloped countries, because of regional and economic reasons. Economically, aminoglycoside drugs are very inexpensive to produce, which is of greatest importance to powerless countries . In addition, aminoglycosides are often prescribed indispensably as a first-line therapy in multidrug-resistant bacteria in many types of infections especially tuberculosis disease (TB), which is still a very serious problem worldwide. The World Health Organization declared that TB is the second leading cause of death from a single infectious agent in the world and continues to be one of the world’s major infectious diseases . Despite the incidence of TB stabilizing in recent years, an estimated one-third of the world’s population remain infected, resulting in over 9 million cases of currently active disease and approximately 1.3 million deaths per year . Tuberculosis cases are still more common in Asian and African (85% of the cases) countries . Therefore, the prescription of aminoglycoside is probably rather high in these countries.
The objective of this work was to assess the caffeic acid phenethyl ester (CAPE) with antioxidant properties in the prevention or attenuation of ototoxicity caused by long-term aminoglycoside administration in a rat model. Caffeic acid phenethyl ester is one of the major components of honeybee propolis extracts. It has been used in traditional medicine for many years as a pharmacologically active and safe compound with anti-inflammatory, antiviral, antimitogenic, anticarcinogenic, immunomodulatory, and antioxidant effects . It has been already shown that CAPE administration has protective effects against oxidative damage caused by different agents in various tissues such as kidney, liver, heart, lung, red blood cell, brain, and neural structures in rat models . But, to our knowledge, the efficacy of CAPE against the toxic effects of aminoglycoside in the inner ear has not been investigated yet. In the present study, we examined the auditory and histopathological changes in cochlea exposed to aminoglycoside with/without CAPE.
2
Materials and methods
All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council . The study was also approved by the Ethical Committee of our institution under permit 2011/27.
2.1
Experimental design
Thirty-two male adult Wistar albino rats weighing between 185 and 295 g were used in this study. They were maintained according to the standard guidelines. All animals were housed reasonably in cages under standard environmental conditions (room temperature between 22°C and 24°C and 50% relative humidity within a 12-hour light/12-hour dark cycle photoperiod). All the animals had free access to water and conventional laboratory diet until before sacrifice. Animals were ranked by weight at the beginning of the study to ensure similar starting body masses between groups. They were divided into 4 experimental groups: control group (n = 8), streptomycin-treated group (n = 8), CAPE-treated group (n = 8), and streptomycin + CAPE–treated group (n = 8). Saline (2.5 mL/kg, intramuscularly) was used in the control group. In the other groups; streptomycin, CAPE, and streptomycin + CAPE were used for 45 days until surgery. Streptomycin was administered via the intramuscular way (dose of 20 mg/[kg d]). Caffeic acid phenethyl ester was purchased from SIGMA (Sigma-Aldrich Co LLC, St Louis, MO) and intraperitoneally injected once a day at a dose of 10 μ mol/kg. According to a previous report, at a concentration of 10 μ mol/L, CAPE completely inhibits the production of reactive oxygen species . Another report suggested that the antioxidant activity of CAPE is dose dependent . Because of these reasons, throughout the entire study without interruption, 10 μ mol/L CAPE was administered every day. The first dose of CAPE was given 24 hours before streptomycin administration and continued until sacrifice.
2.2
Anesthesia
Rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar, Pfizer, Istanbul, Turkey) 60 mg/kg and 2% xylazine hydrochloride (Rompun, Bayer, Istanbul, Turkey) 10 mg/kg by intramuscular injection before evaluating the hearing and before sacrifice.
2.3
Hearing assessment
To test the integrity of the hair cells, all animals were tested with distortion product otoacoustic emissions (DPOAEs) under anesthesia in a quiet room (less than 50 dB background noise). Before the DPOAE was measured, otoscopy was performed to confirm that the external auditory canal and tympanic membrane were normal. Only rats with normal ear canal and tympanic membrane and with initial otoacoustic examination showing normal responses were included in this study. The OAE recordings were elicited from the right and left ear of each animal using a standard commercial ILO-96 OAE apparatus cochlear emission analyzer (Otodynamics Ltd, London, UK). The data were processed and evaluated with otoacoustic emission (OAE) software (EZ Screen 2 Otodynamics OAE Screening and Data Management Software, Hatfield, UK). Each DPOAE test required about 3 minutes to perform. The DPOAEs were recorded before drug administration at the first day and before the animals were killed. Following anesthesia, the primary tones were introduced into the animals’ outer ear canal through an inserted earphone using a plastic adapter that sealed the probe in the outer ear canal. Equilevel primary tones f1 (65 dB) and f2 (55 dB) were fixed at f1/f2 = 1.22, and DPOAEs were measured at 5 different frequencies ranging from 2000 to 8000 Hz (2002, 3003, 4004, 6006, and 8008 Hz). The DPOAEs were determined as DPgrams. The baseline hearing status of all animals was determined with DPgram, and the signal-to-noise ratio (SNR) was found. For each animal, SNRs at 5 frequencies were recorded. Data were collected separately for each rat, and the results were statistically analyzed.
2.4
Operation procedure
All procedures were performed under clean but nonsterile conditions. After being anesthetized, they were decapitated. After fixation by intralabyrinthine perfusion of 4% paraformaldehyde (pH 7.4), the cochleas were removed. The skull bone of the subjects was cut in the midline, and each temporal bone was dissected by scalpel and a pair of scissors. Using the hands and having the external auditory canal as a guide, the bulla was localized with the thumbs. Their temporal bones containing the tympanic bulla were separated from other structures to reveal the cochlea by holding it with one hand and with a hemostatic clamp. The bulla was opened by holding it with one hand; and with a hemostatic clamp, an opening on the posterior air sinus (mastoid) was made. Then, positioning the clamp in the external auditory canal, in a single movement, all bone parts of the leaflet were broken, exposing the cochlea. In the end, the cochleas were dissected and removed en bloc. Then, the cochleas were fixed into a 10% formaldehyde solution and stored for immunohistochemical study.
2.5
Tissue preparation
The cochleas of each rat were fixed for 24 hours in 10% formaldehyde solution and subjected to decalcification for 3 weeks in 10% ethylenediamine tetraacetic acid solution. After the fixation and decalcification processes, the cochleas were washed with tap water for 24 hours, were dehydrated by reaction with graded alcohol series, and were transparented and blocked after infiltration with paraffin. Each paraffin-embedded specimen was sectioned with a microtome (Leica RM 2125, Leica Microsystems, Nussloch GmbH, Germany) at a thickness of 5 μm. Each section was stained with hematoxylin and eosin (H&E) solution and observed with a Nikon ECLIPSE 80i (Japan) microscope. On the other hand, the remaining tissue sections were deparaffinized and washed after reaction with alcohol series and after incubation with 0.1% to 1% H 2 O 2 , washed with phosphate-buffered saline (PBS), and dyed with avidin-biotin. The sections were conducted with 10% normal bovine serum. Then, caspase-3 rat polyclonal IgG primary antibody was diluted with 1:400 normal bovine serum. The sections were incubated with caspase-3 during the night. Phosphate-buffered saline was dripped onto negative control sections. Next day, the sections were washed with PBS and incubated firstly with secondary antibody (biotin bovine antirat) and then horseradish peroxidase and, after washing with PBS, conducted with diaminobenzidine chromogen. The sections washed with distilled water were dyed by hemotoxylin. Then, the sections were washed with distilled water until blueness disappeared, and closed after reaction with alcohol and xylene. The sections were evaluated according to the intensity of caspase-3 immunoreaction under light microscope (Nikon ECLIPSE 80i).
2.6
Statistical analysis
Statistical evaluation was carried out using SPSS 15.0 version for Windows (SPSS Inc, Chicago, IL). Results were analyzed statistically by Kruskal-Wallis to determine differences in amplitudes of DPOAEs and corresponding noise floor differences and thresholds for each frequency. The histopathological variations of streptomycin, CAPE, and streptomycin with CAPE and without medication (control) were evaluated and compared with each other by Mann-Whitney U test. Statistically significant level was accepted as P value less than .05.
2
Materials and methods
All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council . The study was also approved by the Ethical Committee of our institution under permit 2011/27.
2.1
Experimental design
Thirty-two male adult Wistar albino rats weighing between 185 and 295 g were used in this study. They were maintained according to the standard guidelines. All animals were housed reasonably in cages under standard environmental conditions (room temperature between 22°C and 24°C and 50% relative humidity within a 12-hour light/12-hour dark cycle photoperiod). All the animals had free access to water and conventional laboratory diet until before sacrifice. Animals were ranked by weight at the beginning of the study to ensure similar starting body masses between groups. They were divided into 4 experimental groups: control group (n = 8), streptomycin-treated group (n = 8), CAPE-treated group (n = 8), and streptomycin + CAPE–treated group (n = 8). Saline (2.5 mL/kg, intramuscularly) was used in the control group. In the other groups; streptomycin, CAPE, and streptomycin + CAPE were used for 45 days until surgery. Streptomycin was administered via the intramuscular way (dose of 20 mg/[kg d]). Caffeic acid phenethyl ester was purchased from SIGMA (Sigma-Aldrich Co LLC, St Louis, MO) and intraperitoneally injected once a day at a dose of 10 μ mol/kg. According to a previous report, at a concentration of 10 μ mol/L, CAPE completely inhibits the production of reactive oxygen species . Another report suggested that the antioxidant activity of CAPE is dose dependent . Because of these reasons, throughout the entire study without interruption, 10 μ mol/L CAPE was administered every day. The first dose of CAPE was given 24 hours before streptomycin administration and continued until sacrifice.
2.2
Anesthesia
Rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar, Pfizer, Istanbul, Turkey) 60 mg/kg and 2% xylazine hydrochloride (Rompun, Bayer, Istanbul, Turkey) 10 mg/kg by intramuscular injection before evaluating the hearing and before sacrifice.
2.3
Hearing assessment
To test the integrity of the hair cells, all animals were tested with distortion product otoacoustic emissions (DPOAEs) under anesthesia in a quiet room (less than 50 dB background noise). Before the DPOAE was measured, otoscopy was performed to confirm that the external auditory canal and tympanic membrane were normal. Only rats with normal ear canal and tympanic membrane and with initial otoacoustic examination showing normal responses were included in this study. The OAE recordings were elicited from the right and left ear of each animal using a standard commercial ILO-96 OAE apparatus cochlear emission analyzer (Otodynamics Ltd, London, UK). The data were processed and evaluated with otoacoustic emission (OAE) software (EZ Screen 2 Otodynamics OAE Screening and Data Management Software, Hatfield, UK). Each DPOAE test required about 3 minutes to perform. The DPOAEs were recorded before drug administration at the first day and before the animals were killed. Following anesthesia, the primary tones were introduced into the animals’ outer ear canal through an inserted earphone using a plastic adapter that sealed the probe in the outer ear canal. Equilevel primary tones f1 (65 dB) and f2 (55 dB) were fixed at f1/f2 = 1.22, and DPOAEs were measured at 5 different frequencies ranging from 2000 to 8000 Hz (2002, 3003, 4004, 6006, and 8008 Hz). The DPOAEs were determined as DPgrams. The baseline hearing status of all animals was determined with DPgram, and the signal-to-noise ratio (SNR) was found. For each animal, SNRs at 5 frequencies were recorded. Data were collected separately for each rat, and the results were statistically analyzed.
2.4
Operation procedure
All procedures were performed under clean but nonsterile conditions. After being anesthetized, they were decapitated. After fixation by intralabyrinthine perfusion of 4% paraformaldehyde (pH 7.4), the cochleas were removed. The skull bone of the subjects was cut in the midline, and each temporal bone was dissected by scalpel and a pair of scissors. Using the hands and having the external auditory canal as a guide, the bulla was localized with the thumbs. Their temporal bones containing the tympanic bulla were separated from other structures to reveal the cochlea by holding it with one hand and with a hemostatic clamp. The bulla was opened by holding it with one hand; and with a hemostatic clamp, an opening on the posterior air sinus (mastoid) was made. Then, positioning the clamp in the external auditory canal, in a single movement, all bone parts of the leaflet were broken, exposing the cochlea. In the end, the cochleas were dissected and removed en bloc. Then, the cochleas were fixed into a 10% formaldehyde solution and stored for immunohistochemical study.
2.5
Tissue preparation
The cochleas of each rat were fixed for 24 hours in 10% formaldehyde solution and subjected to decalcification for 3 weeks in 10% ethylenediamine tetraacetic acid solution. After the fixation and decalcification processes, the cochleas were washed with tap water for 24 hours, were dehydrated by reaction with graded alcohol series, and were transparented and blocked after infiltration with paraffin. Each paraffin-embedded specimen was sectioned with a microtome (Leica RM 2125, Leica Microsystems, Nussloch GmbH, Germany) at a thickness of 5 μm. Each section was stained with hematoxylin and eosin (H&E) solution and observed with a Nikon ECLIPSE 80i (Japan) microscope. On the other hand, the remaining tissue sections were deparaffinized and washed after reaction with alcohol series and after incubation with 0.1% to 1% H 2 O 2 , washed with phosphate-buffered saline (PBS), and dyed with avidin-biotin. The sections were conducted with 10% normal bovine serum. Then, caspase-3 rat polyclonal IgG primary antibody was diluted with 1:400 normal bovine serum. The sections were incubated with caspase-3 during the night. Phosphate-buffered saline was dripped onto negative control sections. Next day, the sections were washed with PBS and incubated firstly with secondary antibody (biotin bovine antirat) and then horseradish peroxidase and, after washing with PBS, conducted with diaminobenzidine chromogen. The sections washed with distilled water were dyed by hemotoxylin. Then, the sections were washed with distilled water until blueness disappeared, and closed after reaction with alcohol and xylene. The sections were evaluated according to the intensity of caspase-3 immunoreaction under light microscope (Nikon ECLIPSE 80i).
2.6
Statistical analysis
Statistical evaluation was carried out using SPSS 15.0 version for Windows (SPSS Inc, Chicago, IL). Results were analyzed statistically by Kruskal-Wallis to determine differences in amplitudes of DPOAEs and corresponding noise floor differences and thresholds for each frequency. The histopathological variations of streptomycin, CAPE, and streptomycin with CAPE and without medication (control) were evaluated and compared with each other by Mann-Whitney U test. Statistically significant level was accepted as P value less than .05.
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