Abstract
Objectives
Sildenafil, a selective inhibitor of phosphodiesterase type 5, is widely used for the treatment of erectile dysfunction. Although cochlear effects of phosphodiesterase type 5 inhibitors remain still unclear because of inadequate data, some evidence that recently emerged indicates that these medications may be responsible for hearing impairment. In the present study, we aimed to examine the histopathologic effects of long-term sildenafil use on the cochlea in a rat model.
Methods
The study was performed with adult male Wistar albino rats. The control group was fed on standard laboratory diet. The study group was applied orally with sildenafil therapy, 1.5 mg/kg once a day for 45 days. Rats were anesthetized and decapitated. Each temporal bone was dissected, and the cochleas were removed en bloc. The inner-ear biopsy specimens were examined histologically with hematoxylin and eosin and caspase 3 immunoreaction under light microscopy.
Results
Hematoxylin and eosin staining showed no distinctive difference between the control group and the sildenafil group. With immunohistochemical examination, caspase 3 immunoreactivity was observed in the sildenafil group. In the control group, caspase 3 immunoreactivity was not observed.
Conclusions
The caspase 3 immunoreactivity in the sildenafil group was strongly associated with an increase in apoptotic events in the cochlea. Long-term use of sildenafil can cause hearing impairment through increased apoptosis.
1
Introduction
Ototoxic drugs can cause alterations in balance and hearing functions by modifying the cochlear and vestibular system. The most common drugs implicated as having ototoxic side effects are mainly aminoglycoside antibiotics; some chemotherapy agents especially platinum-based antineoplastic drugs (cisplatin and carboplatin); and some loop diuretics such as furosemide, salicylates, and quinine . Although there are no adequate and reliable data yet, some recently emerged evidence indicates that phosphodiesterase type 5 (PDE-5) inhibitors may also be responsible for hearing impairment .
Phosphodiesterase type 5 inhibitors—sildenafil; vardenafil; tadalafil; and several newly developed drugs such as udenafil, lodenafil, mirodenafil, and others—are prescription drugs that are taken orally to treat erectile dysfunction (ED), but sildenafil is also indicated for the treatment of pulmonary arterial hypertension (PAH) . Sildenafil was the first PDE-5 inhibitor licensed for ED, and it has been prescribed widely with good tolerance since approved by the US Food and Drug Administration (FDA) in 1998. Sildenafil is also indicated for the treatment of PAH since approved in 2005 . Sildenafil, although intermittently used, sometimes must be taken continually in high doses. For the treatment of ED, sildenafil citrate is widely administered as needed, and the recommended dose is 25 to 100 mg daily . On the other hand, sildenafil is used in both continuous and high doses in patients with PAH . The recommended dose of sildenafil for PAH is 20 mg, 3 times a day (60 mg/d) .
Although sildenafil has been well tolerated in clinical practice largely since more than a decade, recently, some reports of various adverse events, usually mild and temporary, have been described at varying rates. The most commonly reported adverse effects associated with sildenafil treatment in the literature were headache, facial flushing, dyspepsia, myalgia, visual disturbances, and nasal congestion; less encountered side effects were heartburn; nausea; dry mouth; diarrhea; rash; lowered blood pressure; reduction in olfactory sensitivity; and neurologic and emotional disturbances such as dizziness, depression, insomnia, abnormal dreams, amnesia, loss of consciousness, and increased aggressive behavior . It was not until 2007 that a case of hearing loss (HL) was reported as a potential side effect of sildenafil . Then, sildenafil has been implicated as a causative agent in hearing impairment, and there has been an increasing interest in the potential of HL with PDE-5 inhibitors . A cross-sectional epidemiological study to evaluate the association between PDE-5 inhibitors use and HL supported that current findings regarding the risk of HL related to PDE-5 inhibitors use seem to be justified . Contrary to this, some authors argued that sildenafil has a good safety profile and does not create adverse affects on hearing system .
It seems that the cochlear effect of using sildenafil is still unclear. So far, there are only a few studies that investigated the relation between PDE-5 inhibitor use and HL . These are mostly case reports and a small number of epidemiological and audiologic assessments. To our knowledge, no study to date have investigated the histopathologic changes in the inner ear caused by the use of sildenafil. In present study, we aimed to evaluate the effect of long-term sildenafil use on cochlea, histopathologically. In addition to histopathologic examination with hematoxylin and eosin (H&E) staining, caspase 3 was also used for immunohistochemical examination because caspase 3 is more effective than H&E staining to distinguish apoptotic cells.
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 .
2.1
Animals and treatment
Twenty male 5-month-old Wistar albino rats weighing between 225 and 285 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). Mice 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 2 experimental groups. Group 1 (n = 10) was control group, and any medication was not applied in this group. Group 2 (n = 10) was sildenafil group. Sildenafil was obtained from 50 mg Viagra tablets (Viagra; Pfizer, Istanbul, Turkey), which were ground into fine powder and dissolved in 0.9% NaCl. This process was repeated daily for 45 days. The sildenafil citrate dose administered in this study was 1.5 mg/kg body weight . This dose is compatible with the per os doses normally used in humans, without appreciable dose-dependent variations. Sildenafil therapy was applied orally with orogastric tube once a day for 45 days until 12 hours before surgery.
To evaluate the hearing, transient otoacoustic emissions were planned and started. However, after a couple days, because of unexpected mechanical problems, it was not possible to complete the measurement of hearing thresholds.
2.2
Operation procedure
All procedures were performed under clean but nonsterile conditions. Each rat was anesthetized with combination of ketamine hydrochloride (Ketalar) 60 mg/kg and 2% xylazine hydrochloride (Rompun) 10 mg/kg by intramuscular injection. After being anesthesized, they were decapitated. The skull bones of the subjects were 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 bullas were separated from other structures to reveal the cochleae 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, by positioning the clamp in the external auditory canal, in a single movement, the bone part of the leaflet was broken, exposing the cochlea. At the end, the cochleas were dissected and removed en bloc. Biopsy specimens from the inner ear were fixed into a 10% formaldehyde solution and stored for histopathologic examinations. Examination was performed by experienced pathologists in blinded fashion.
2.3
Tissue preparation
The cochleas of each rat were fixed for 24 hours in 10% formaldehyde solution, and the cochleas were conducted to decalcification for 3 weeks in 10% EDTA solution. After 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 being infiltrated with paraphine. The tissue sectioned with a microtome (Microm HM 360, Walldorf, Germany) in slices of 5- μ m thickness, and the biopsy specimens from inner ears were stained with H&E. On the other hand, remaining tissue sections were deparaphinized and washed after being reacted with alcohol series, and after incubation with 0.1% to 1% H 2 O 2 , they were washed with phosphate-buffered saline (PBS) and dyed with avitin biotine. The sections were conducted with 10% normal bovine serum. Then, caspase 3 rat polyclonal immunoglobulin G 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 into negative control sections. On the next day, the sections were washed with PBS and firstly incubated with secondary antibody (biotin bovine antirat) then horseradish peroxidase, and after being washed with PBS, they were conducted with 3,3′-Diaminobenzidine chromogen. The sections washed with distilled water were dyed by hemotoxylin; then, the sections were washed with distilled water until blueness disappeared and were closed after being reacted with alcohol and xylene. The sections were evaluated according to the intensity of caspase 3 immunoreaction under light microscope (Nikon ECLIPSE 80i, Japan).
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 .
2.1
Animals and treatment
Twenty male 5-month-old Wistar albino rats weighing between 225 and 285 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). Mice 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 2 experimental groups. Group 1 (n = 10) was control group, and any medication was not applied in this group. Group 2 (n = 10) was sildenafil group. Sildenafil was obtained from 50 mg Viagra tablets (Viagra; Pfizer, Istanbul, Turkey), which were ground into fine powder and dissolved in 0.9% NaCl. This process was repeated daily for 45 days. The sildenafil citrate dose administered in this study was 1.5 mg/kg body weight . This dose is compatible with the per os doses normally used in humans, without appreciable dose-dependent variations. Sildenafil therapy was applied orally with orogastric tube once a day for 45 days until 12 hours before surgery.
To evaluate the hearing, transient otoacoustic emissions were planned and started. However, after a couple days, because of unexpected mechanical problems, it was not possible to complete the measurement of hearing thresholds.
2.2
Operation procedure
All procedures were performed under clean but nonsterile conditions. Each rat was anesthetized with combination of ketamine hydrochloride (Ketalar) 60 mg/kg and 2% xylazine hydrochloride (Rompun) 10 mg/kg by intramuscular injection. After being anesthesized, they were decapitated. The skull bones of the subjects were 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 bullas were separated from other structures to reveal the cochleae 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, by positioning the clamp in the external auditory canal, in a single movement, the bone part of the leaflet was broken, exposing the cochlea. At the end, the cochleas were dissected and removed en bloc. Biopsy specimens from the inner ear were fixed into a 10% formaldehyde solution and stored for histopathologic examinations. Examination was performed by experienced pathologists in blinded fashion.
2.3
Tissue preparation
The cochleas of each rat were fixed for 24 hours in 10% formaldehyde solution, and the cochleas were conducted to decalcification for 3 weeks in 10% EDTA solution. After 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 being infiltrated with paraphine. The tissue sectioned with a microtome (Microm HM 360, Walldorf, Germany) in slices of 5- μ m thickness, and the biopsy specimens from inner ears were stained with H&E. On the other hand, remaining tissue sections were deparaphinized and washed after being reacted with alcohol series, and after incubation with 0.1% to 1% H 2 O 2 , they were washed with phosphate-buffered saline (PBS) and dyed with avitin biotine. The sections were conducted with 10% normal bovine serum. Then, caspase 3 rat polyclonal immunoglobulin G 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 into negative control sections. On the next day, the sections were washed with PBS and firstly incubated with secondary antibody (biotin bovine antirat) then horseradish peroxidase, and after being washed with PBS, they were conducted with 3,3′-Diaminobenzidine chromogen. The sections washed with distilled water were dyed by hemotoxylin; then, the sections were washed with distilled water until blueness disappeared and were closed after being reacted with alcohol and xylene. The sections were evaluated according to the intensity of caspase 3 immunoreaction under light microscope (Nikon ECLIPSE 80i, Japan).
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