Abstract
Objective
The aim of the study was to investigate the protective effects of Salvia miltiorrhiza (SM) against cisplatin-induced ototoxicity in guinea pigs.
Methods
Thirty-nine guinea pigs were randomly divided into 3 groups. The first group (control group) received physiologic saline by intraperitoneal (i.p.) injection for 5 days. The second group (cisplatin group) was treated with cisplatin (2 mg/kg per day, i.p. injection) for 5 days. The third group (SM group) was given SM (8 g/kg per day, i.p. injection) for 2 days and then was given SM (8 g/kg per day, i.p. injection) and cisplatin (2 mg/kg per day, i.p. injection) for 5 days. Auditory brain stem response (ABR) and cochlea blood flow measurement were used to evaluate cochlea function. The structures of cochlea were observed by light microscope, scanning electron microscope, transmission electron microscope (TEM), and immunohistochemical examination.
Results
Cisplatin could cause severe acoustic damages including significant elevation of ABR threshold, substantial losses of outer hair cells and inner hair cells, and severe damage on the stria vascularis and spiral ganglion cells (SGCs). Although in SM group, the increased tendency of threshold was milder than that in cisplatin group. The damages in cochlea and stria vascularis were also less severe than those in cisplatin group. The expression of induced nitric oxide synthase in the cochlea and SGC in SM group was lower than that in cisplatin group.
Conclusions
Salvia miltiorrhiza can significantly reduce the cisplatin-induced side effects.
1
Introduction
Cisplatin has been extensively used as an antineoplastic agent to treat a variety of cancers. The 2 commonly reported dose-limiting side effects of cisplatin are nephrotoxicity and ototoxicity. Although nephrotoxicity can be controlled with hydration therapy, ototoxicity still poses a limitation to effective cisplatin chemotherapy. The exact mechanism involved in cisplatin-induced ototoxicity remains obscure. A great deal of evidence has demonstrated that cisplatin ototoxicity may result from free radical-mediated oxidative damages . Cisplatin increases the activity of induced nitric oxide synthase (iNOS), and iNOS catalyzed large amounts of nitric oxide (NO) that takes part in the cytotoxicity of cisplatin . In the cardiovascular system, NO acts as an endothelium-derived relaxing factor, regulating blood flow through its action on the smooth muscle cells of blood vessels. Induced nitric oxide synthase catalyzes 100 to 1000-fold amounts of NO. In cochlea, iNOS activities are also under pathologic conditions, for example, inflammation , noise exposure , endolymphatic hydrops , or administration of ototoxic drugs. The NOS inhibitor reduces the functional disturbance of cochlea. Salvia miltiorrhiza , as a traditional Chinese medicine, can improve microcirculation and eliminate free radical. Thus, it was widely used in medical field to treat many otolaryngology diseases, such as sudden deafness , presbycusis , noise-induced deafness , and aminoglycoside antibiotic-induced deafness . The purpose of this study was to investigate the effect of Salvia miltiorrhiza on ototoxicity induced by cisplatin.
2
Materials and methods
2.1
Experiment I
2.1.1
Treatment of animals with cisplatin in the presence or absence of pretreatment with Salvia miltiorrhiza
Thirty-nine guinea pigs weighing 300 to 400 g with normal auricular reflex were used in this study and were randomly divided into 3 groups (13 guinea pigs in each group). The first group (control group) received physiologic saline without cisplatin by intraperitoneal (i.p.) injection for 5 days. The second group (cisplatin group) was treated with cisplatin (2 mg/kg per day, i.p. injection) for 5 days. The third group ( Salvia miltiorrhiza group) was given Salvia miltiorrhiza (8 g/kg per day, i.p. injection) for 2 days and then was given Salvia miltiorrhiza (8 g/kg per day, i.p. injection) and cisplatin (2 mg/kg per day, i.p. injection) for 5 days. After injection finished, 3 guinea pigs were killed for stria vascularis surface preparation, 2 guinea pigs were killed for scanning electron microscope (SEM) observation, 2 guinea pigs were killed for transmission electron microscope (TEM) observation, and 6 guinea pigs were killed for iNOS detection. All of the animals were approved by the Laboratory Animal Care of Hebei Medical University (Hebei Province, China). Animals were cared for and killed according to the principles of animal care outlined by the Chinese Academy of Sciences. All efforts were made to use the minimal number of animals in these studies.
2.1.2
Body weight
All the animals were weighed before the first treatment and 24 hours after the final treatment.
2.1.3
Auditory brain stem response measurement
Auditory brain stem response (ABR) threshold were measured by ABR equipment (San-ei TS 12 type, Tokyo, Japan) before the first treatment and 24 hours after the final treatment. Guinea pigs were anesthetized with 100 mg/kg of ketamine hydrochloride injection (i.p.). The reference electrode was inserted subcutaneously below the ipsilateral right ear, and the stimulating electrode was inserted at the vertex. The ground electrode was located on the top of the nose. The TDH-39 type earphone delivered sound, which was 1 cm away from the tested external ear canal. The reaction had been drawn from the electrode and enlarged as a result of preposed amplifier. After computer processing, the result was displayed on the computer screen. Waves III was used in ABR measurement.
2.1.4
Cochlea blood flow measurement
After animals had been measured with ABR threshold, their skin and muscle tissue were sheared off from the posterior ear to expose the otic vesicle. The otic vesicle was opened, and the basement turn of the cochlea was exposed, of which some part of mucoperiosteum was peeled under microscope (CX21FS1; Olympus, Optical Co Ltd, Japan).With the help of 3-dimensional thruster periflux PF3 (Periflux PF3; Perimed, Stockholm, Sweden), probe (302 type, 0.6-mm diameter) was fixed on the eminence of the cochlea basal turn (promontory). The signal was output into blood stream recorder (Servogor 120; Goerz Company, Vienna, Austria), and blood flow was recorded for 3 minutes.
2.2
Experiment II: morphological studies
One day after the final injection, the animals were decapitated. Otic vesicle was removed immediately, and the cochlea was fixed with different fixatives, including 4% paraformaldehyde, 2.5% glutaraldehyde, and 4% glutaraldehyde. The specimens were prepared by use of different treatments for light microscope (LM), SEM, and TEM, respectively.
2.2.1
Stria vascularis surface
The cochlea was fixed with 4% paraformaldehyde after the blood flushing out with 0.1 mol/L phosphate-buffered saline. The bony capsule was removed, and the lateral wall tissues (spiral ligament and stria vascularis) were separated. Only the stria vascularis from the second and basal turns were processed further and mounted with neutral resin around the species. The structure of capillaries was observed under LM.
2.2.2
Preparation of SEM
The cochlea was fixed with 2.5% glutaraldehyde for 4 to approximately 6 hours. The bony capsule was removed, and the basilar membrane was dehydrated, dried, gilded, and observed under SEM (S-3500 type; Hitachi, Tokyo, Japan).
2.2.3
Preparation of TEM
The cochlea was fixed with 4% glutaraldehyde for 4 to approximately 6 hours and decalcified in 10% EDTA for 10 days. The basilar membrane and spiral ligament were rinsed, postfixed with osmium acid, dehydrated with a graded series of ethanol, and embedded in resin. Serial midmodiolar sections were cut at 50-nm thickness, stained, and observed under TEM (H-7500 type; Hitachi, Tokyo, Japan).
2.2.4
Detection of iNOS
The cochlea was fixed with 4% paraformaldehyde, immersed in the same fixative for 1 week, decalcified in 10% EDTA for 2 to approximately 3 weeks, dehydrated, embedded in paraffin, and sliced. The paraffin was removed, and the sections were dealt with immunohistochemical methods (Wuhan Boster Biological Technology, Wuhan, China). Induced nitric oxide synthase positive expression of outer hair cell (OHC), inner hair cell (IHC), and spiral ganglion cells (SGCs) in cochlea base turn was evaluated by gray value that was measured by Luzex-F Image Analysis Software (Nireco, Tokyo, Japan). Higher gray value means lower degree of iNOS expression.
2.3
Statistics
The data were expressed as mean ± SD. The weight data were analyzed by paired t test with P < .05 as a level of significance. After treatment, q test of analysis variance was used when comparing ABR threshold, cochlea blood flow, and gray value of iNOS in each group with P < .05 as a level of significance.
2
Materials and methods
2.1
Experiment I
2.1.1
Treatment of animals with cisplatin in the presence or absence of pretreatment with Salvia miltiorrhiza
Thirty-nine guinea pigs weighing 300 to 400 g with normal auricular reflex were used in this study and were randomly divided into 3 groups (13 guinea pigs in each group). The first group (control group) received physiologic saline without cisplatin by intraperitoneal (i.p.) injection for 5 days. The second group (cisplatin group) was treated with cisplatin (2 mg/kg per day, i.p. injection) for 5 days. The third group ( Salvia miltiorrhiza group) was given Salvia miltiorrhiza (8 g/kg per day, i.p. injection) for 2 days and then was given Salvia miltiorrhiza (8 g/kg per day, i.p. injection) and cisplatin (2 mg/kg per day, i.p. injection) for 5 days. After injection finished, 3 guinea pigs were killed for stria vascularis surface preparation, 2 guinea pigs were killed for scanning electron microscope (SEM) observation, 2 guinea pigs were killed for transmission electron microscope (TEM) observation, and 6 guinea pigs were killed for iNOS detection. All of the animals were approved by the Laboratory Animal Care of Hebei Medical University (Hebei Province, China). Animals were cared for and killed according to the principles of animal care outlined by the Chinese Academy of Sciences. All efforts were made to use the minimal number of animals in these studies.
2.1.2
Body weight
All the animals were weighed before the first treatment and 24 hours after the final treatment.
2.1.3
Auditory brain stem response measurement
Auditory brain stem response (ABR) threshold were measured by ABR equipment (San-ei TS 12 type, Tokyo, Japan) before the first treatment and 24 hours after the final treatment. Guinea pigs were anesthetized with 100 mg/kg of ketamine hydrochloride injection (i.p.). The reference electrode was inserted subcutaneously below the ipsilateral right ear, and the stimulating electrode was inserted at the vertex. The ground electrode was located on the top of the nose. The TDH-39 type earphone delivered sound, which was 1 cm away from the tested external ear canal. The reaction had been drawn from the electrode and enlarged as a result of preposed amplifier. After computer processing, the result was displayed on the computer screen. Waves III was used in ABR measurement.
2.1.4
Cochlea blood flow measurement
After animals had been measured with ABR threshold, their skin and muscle tissue were sheared off from the posterior ear to expose the otic vesicle. The otic vesicle was opened, and the basement turn of the cochlea was exposed, of which some part of mucoperiosteum was peeled under microscope (CX21FS1; Olympus, Optical Co Ltd, Japan).With the help of 3-dimensional thruster periflux PF3 (Periflux PF3; Perimed, Stockholm, Sweden), probe (302 type, 0.6-mm diameter) was fixed on the eminence of the cochlea basal turn (promontory). The signal was output into blood stream recorder (Servogor 120; Goerz Company, Vienna, Austria), and blood flow was recorded for 3 minutes.
2.2
Experiment II: morphological studies
One day after the final injection, the animals were decapitated. Otic vesicle was removed immediately, and the cochlea was fixed with different fixatives, including 4% paraformaldehyde, 2.5% glutaraldehyde, and 4% glutaraldehyde. The specimens were prepared by use of different treatments for light microscope (LM), SEM, and TEM, respectively.
2.2.1
Stria vascularis surface
The cochlea was fixed with 4% paraformaldehyde after the blood flushing out with 0.1 mol/L phosphate-buffered saline. The bony capsule was removed, and the lateral wall tissues (spiral ligament and stria vascularis) were separated. Only the stria vascularis from the second and basal turns were processed further and mounted with neutral resin around the species. The structure of capillaries was observed under LM.
2.2.2
Preparation of SEM
The cochlea was fixed with 2.5% glutaraldehyde for 4 to approximately 6 hours. The bony capsule was removed, and the basilar membrane was dehydrated, dried, gilded, and observed under SEM (S-3500 type; Hitachi, Tokyo, Japan).
2.2.3
Preparation of TEM
The cochlea was fixed with 4% glutaraldehyde for 4 to approximately 6 hours and decalcified in 10% EDTA for 10 days. The basilar membrane and spiral ligament were rinsed, postfixed with osmium acid, dehydrated with a graded series of ethanol, and embedded in resin. Serial midmodiolar sections were cut at 50-nm thickness, stained, and observed under TEM (H-7500 type; Hitachi, Tokyo, Japan).
2.2.4
Detection of iNOS
The cochlea was fixed with 4% paraformaldehyde, immersed in the same fixative for 1 week, decalcified in 10% EDTA for 2 to approximately 3 weeks, dehydrated, embedded in paraffin, and sliced. The paraffin was removed, and the sections were dealt with immunohistochemical methods (Wuhan Boster Biological Technology, Wuhan, China). Induced nitric oxide synthase positive expression of outer hair cell (OHC), inner hair cell (IHC), and spiral ganglion cells (SGCs) in cochlea base turn was evaluated by gray value that was measured by Luzex-F Image Analysis Software (Nireco, Tokyo, Japan). Higher gray value means lower degree of iNOS expression.
2.3
Statistics
The data were expressed as mean ± SD. The weight data were analyzed by paired t test with P < .05 as a level of significance. After treatment, q test of analysis variance was used when comparing ABR threshold, cochlea blood flow, and gray value of iNOS in each group with P < .05 as a level of significance.
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