Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation.

Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients.

Ototoxicity.

This paper reviews intriguing recent findings on the mechanisms of drug induced hearing loss caused by two major classes of therapeutic agents: the aminoglycoside antibiotics and cisplatin. Both drug categories are nephrotoxic as well as ototoxic. Aminoglycosides and cisplatin target the outer hair cells in the basal turn of the cochlea to cause high frequency sensorineural hearing loss in a substantial percentage of patients treated with these drugs. Each group of agents appears to generate reactive oxygen species within the cochlea that trigger downstream mechanisms leading to cell death. Various protective agents including antioxidants show promise in protecting the inner ear from damage in experimental animals. The only successful double-blind, placebo controlled clinical trial using a protective agent to prevent ototoxicity was carried out in China. Aspirin or placebo was given in combination with gentamicin. A significant decrease in hearing loss was observed. Successful clinical implementation of protective agents will require a cautious approach, so that the therapeutic effect of the anti-infective agent or anti-neoplastic drug is not attenuated. This may require novel methods of administration of protective agents, such as injection within the middle ear. This would provide a maximal dose of protective agent without systemic interference with the desired effect of the ototoxic agent.

Expression of the kidney injury molecule 1 in the rat cochlea and induction by cisplatin.

Cisplatin is a widely used chemotherapeutic agent whose dose-limiting side effects include ototoxicity and nephrotoxicity. Recent evidence indicates that cisplatin induces the expression of a novel protein, kidney injury molecule-1, in the renal proximal tubular epithelium to aid in regeneration. In this study, we determined whether kidney injury molecule-1 is expressed in the cochlea and is induced by cisplatin. Using reverse transcriptase polymerase chain reaction techniques, we have now identified kidney injury molecule-1 in the rat cochlea and in three different mouse transformed hair cell lines. Administration of cisplatin to rats produced hearing loss and induced kidney injury molecule-1 mRNA in the rat cochlea. Pretreatment of rats with lipoic acid, a scavenger of reactive oxygen species, significantly reduced cisplatin-induced hearing loss and kidney injury molecule-1 expression. Cisplatin also increased the expression of cochlear NOX3 mRNA, a member of the superoxide generating NADPH oxidase family of proteins recently identified in the cochlea, inhibition of which decreased kidney injury molecule-1 expression. Polymerase chain reaction performed on different regions of the cochlea indicated the presence of kidney injury molecule-1 mRNA in the lateral wall, organ of Corti and spiral ganglion. This distribution was confirmed by immunocytochemistry. Taken together, these data identify kidney injury molecule-1 as a novel cochlear injury molecule, whose expression is regulated by reactive oxygen species generated via the NADPH oxidase pathway.

Time response of carboplatin-induced hearing loss in rat.

Carboplatin is currently being used as an anticancer drug against human cancers. However, high dose of carboplatin chemotherapy resulted in hearing loss in cancer patients. We have shown that carboplatin-induced hearing loss was related to dose-dependent oxidative injury to the cochlea in rat model. However, the time response of ototoxic dose of carboplatin on hearing loss and oxidative injury to cochlea has not been explored. The aim of the study was to evaluate the time response of carboplatin-induced hearing loss and oxidative injury to the cochlea of the rat. Male Wistar rats were divided into two groups of 30 animals each and treated as follows: (1) control (normal saline, i.p.) and (2) carboplatin (256 mg/kg, a single i.p. bolus injection). Auditory brain-evoked responses (ABRs) were recorded before and 1-5 days after treatments. The animals (n = 6) from each group were sacrificed on day 1, 2, 3, 4, and 5 and cochleae were isolated and analyzed. Carboplatin significantly elevated the hearing thresholds to clicks and to 2, 4, 8, 16, and 32 kHz tone burst stimuli only 3-5 days post-treatment. Carboplatin significantly increased nitric oxide (NO), malondialdehyde (MDA) levels and manganese superoxide dismutase (Mn-SOD) activity in the cochlea 4-5 and 3-5 days post-treatment, respectively, indicating enhanced influx of free radicals and oxidative injury to the cochlea. Carboplatin significantly depressed the reduced to oxidized glutathione (GSH/GSSG) ratio, antioxidant enzyme activities such as copper/zinc-superoxide dismutase (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) as well as enzyme protein expressions in the cochlea 3-5 days after treatment. The data suggest that carboplatin-induced hearing loss involves oxidative injury to the cochlea of the rat in a time-dependent manner.

Carboplatin-induced oxidative stress in rat cochlea.

Carboplatin is currently being used in the clinic against a variety of human cancers. However, high dose carboplatin chemotherapy resulted in ototoxicity in cancer patients. This is the first study to show carboplatin-induced oxidative stress response in the cochlea of rat. Male Wistar rats were divided into two groups of six animals each and treated as follows: (1) control (normal saline, i.p.) and (2) carboplatin (256 mg/kg, i.p.). Animals in both groups were sedated with ketamine/xylazine and auditory brainstem-evoked responses were recorded before and 4 days after treatments. The animals were sacrificed on the fourth day and cochleae were harvested and analyzed. A significant elevation of the hearing threshold shifts was noted at clicks, 8, 16, and 32 kHz tone burst stimuli following carboplatin administration. Carboplatin significantly increased nitric oxide and malondialdehyde levels, xanthine oxidase and manganese-superoxide dismutase activities in the cochlea indicating enhanced flux of free radicals. Cochlear glutathione levels, antioxidant enzyme activities such as copper zinc-superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase and enzyme protein levels were significantly depleted 4 days after carboplatin treatment. The data suggest that carboplatin induced free radical generation and antioxidant depletion, and caused oxidative injury in the cochleae of rats.

Pharmacological antagonism of the slow-activating delayed rectifying potassium channel (I(Ks)) has no effect on cochlear structure and function in vivo.

The experimental class III antiarrhythmic drug, L-768673, prolongs the refractory period of cardiac myocytes by selectively blocking the slow-activating delayed rectifying potassium (I(Ks)) channel. The I(Ks) channel has also been identified in vestibular dark cells and in the marginal cells of the stria vascularis. In the stria vascularis, the I(Ks) channel plays an important role in cochlear homeostasis. Genetic null deletion of the I(Ks) channel in mice and man results in profound hearing loss and cochlear pathology. Therefore, the purpose of the present study was to investigate the effect of L-768673 on the auditory function and cochlear morphology in rats using auditory brainstem-evoked response and light microscopy. Auditory testing was performed one week prior to dosing, following 14 days of administration and 28 days after the completion of dosing. L-768673 (50 or 250 mg/kg/day for 14 days), had no significant effects on auditory function or cochlear morphology. The results of this study suggest that high doses of L-768673 are not toxic to the inner ear of adult rats treated for 14 consecutive days, and that the ototoxic potential of orally administered L-768673 and similar I(Ks)-selective compounds is unlikely at doses within the therapeutic range.

Dose response of carboplatin-induced hearing loss in rats: antioxidant defense system.

Carboplatin, a platinum-containing anticancer drug, is currently being used against a variety of cancers. However, a single high dose of carboplatin is ototoxic in cancer patients. This is the first study to show carboplatin-induced hearing loss in a rat model. Male Wistar rats were divided into five groups and treated as follows: (1) control (saline, intraperitoneally (i.p.)); (2) carboplatin (64 mg/kg, i.p.); (3) carboplatin (128 mg/kg i.p.); (4) carboplatin (192 mg/kg, i.p.) and (5) carboplatin (256 mg/kg, i.p.). Animals in all groups were sedated with ketamine/xylazine and auditory brain-evoked responses (ABRs) were recorded before and 4 days after treatments. The animals were sacrificed on the fourth day and cochleae were harvested and analyzed. Carboplatin dose-dependently decreased body weight. However, at higher doses of carboplatin (192 and 256 mg/kg), there was a significant elevation of hearing threshold shifts at clicks, 4, 8, 16 and 32 kHz tone burst stimuli. The higher doses of carboplatin (192 and 256 mg/kg) significantly increased cochlear lipid peroxidation (132 and 146% of control) and depleted cochlear glutathione levels (66 and 63% of control), respectively. The antioxidant enzyme activities such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase (GST) depressed significantly at higher doses of carboplatin. The data suggest that higher doses of carboplatin (above 128 mg/kg) induce hearing loss as evidenced by significant changes in ABRs, lipid peroxidation and antioxidants in the cochlea of rats.

Acute changes in cochlear potentials due to cisplatin.

The purpose of this study was to investigate how the hair cells and stria vascularis are affected at the onset of cisplatin ototoxicity. The effects on the endocochlear potential (EP) and the cochlear microphonics (CM) were observed simultaneously in two groups of adult chinchillas receiving as follows: (1) 5 microl of cisplatin (1 mg/ml) in normal saline, and (2) 5 microl of normal saline on the round window. The EP and the CM were recorded for 12-14 h after cisplatin application, and morphological changes were assessed using scanning electron microscopy. Both the EP and the CM amplitude demonstrated a profound reduction, and a very strong correlation was observed between these two values during this time period. Although the reduction of the EP and the CM was observed by 12-14 h, only very slight degeneration of outer hair cells was seen at that time. These data suggested that a reduction of the EP which was caused by the alteration of the stria vascularis might be primarily responsible for very early changes in cochlear function after topical cisplatin application, while later changes were the direct result of hair cell damage.

Effect of protective agents against cisplatin ototoxicity.

HYPOTHESIS: The goals of this investigation were to compare the efficacy of three protective agents against cisplatin-induced elevation of auditory brainstem response (ABR) thresholds and to examine whether these protective agents prevent cisplatin-induced alterations of the antioxidant defense system in the cochlea of the rat. BACKGROUND: Cisplatin is an ototoxic antitumor agent. Previous animal studies have shown that cisplatin administration causes an elevation of ABR thresholds. These auditory changes are accompanied by alterations in the concentration of glutathione and the antioxidant enzymes in the cochlea. The authors' previous work has indicated that the protective agent diethyldithiocarbamate (DDTC) prevents decrease in glutathione (GSH), alteration of antioxidant enzyme activity, and disruption of cochlear function with cisplatin administration. METHODS: Wistar rats were sedated and underwent pretreatment ABR testing using clicks and tone burst stimuli at 8, 16, and 32 kHz. Control rats received saline by intraperitoneal (i.p.) injection. Positive control rats were administered cisplatin 16 mg/kg i.p. Three groups of rats received protective agents in combination with cisplatin. The DDTC-protected rats were given 600 mg/kg of DDTC subcutaneously 1 hour after cisplatin. Animals protected by 4-methylthiobenzoic acid (MTBA) were given 250 mg/kg of this agent i.p. 30 minutes before cisplatin. Animals protected with ebselen were given 16 mg/kg i.p. one hour before cisplatin. The ABR thresholds were recorded 72 hours after cisplatin administration in all groups. Cochleas were removed, and extracts of the tissues were analyzed for GSH, activities of antioxidant enzymes (superoxide dismutase [SOD], catalase, glutathione peroxidase, and glutathione reductase) and malondialdehyde (MDA) (as an index of lipid peroxidation). RESULTS: Cisplatin-treated rats had significant ABR threshold shifts, ranging from 27 to 40 dB. Rats administered each of the three protective agents in combination with cisplatin had ABR threshold shifts of <10 dB. The cochleae of rats administered cisplatin alone had nearly a 50% depletion of glutathione and about a 50% reduction in the activities of SOD, glutathione peroxidase, and glutathione reductase, while catalase activity was reduced to 70% of control values. These changes were accompanied by a reciprocal elevation of MDA of 165%. These changes, namely, the depletion of GSH and antioxidant enzyme activity and the elevation of MDA in the cochlea, were largely attenuated by the administration of the protective agents tested. CONCLUSION: These findings suggest that cisplatin ototoxicity is related to lipid peroxidation and that the use of protective agents prevents hearing loss and lipid peroxidation by sparing the antioxidant system in the cochlea. These results suggest the possibility that the clinical use of protective agents could effectively reduce or prevent damage to the inner ear of patients receiving cisplatin chemotherapy, provided that the antitumor effect is not altered.

Dose-dependent protection by lipoic acid against cisplatin-induced nephrotoxicity in rats: antioxidant defense system.

This study was designed to investigate the role of graded doses of lipoic acid pretreatment against cisplatin-induced nephrotoxicity. Male Wistar rats were divided into six groups and treated as follows: 1) vehicle (saline) control; 2) cisplatin (16 mg/kg, intraperitoneally); 3) lipoic acid (100 mg/kg, intraperitoneally); 4) cisplatin plus lipoic acid (25 mg/kg); 5) cisplatin plus lipoic acid (50 mg/kg) and 6) cisplatin plus lipoic acid (100 mg/kg). Rats were sacrificed three days after treatment, and plasma as well as kidneys were isolated and analyzed. Plasma creatinine increased (677% of control) following cisplatin administration alone which was decreased by lipoic acid in a dose-dependent manner. Cisplatin-treated rats showed a depletion of renal glutathione (GSH), increased oxidized GSH and decreased GSH/GSH oxidized ratio (62%, 166% and 62% of control), respectively which were restored with lipoic acid pretreatment. Renal superoxide dismutase, catalase, glutathione peroxidase (GSH peroxidase) and glutathione reductase activities decreased (62%, 75%, 62% and 80% of control), respectively, and malondialdehyde content increased (204% of control) following cisplatin administration, which were restored with increasing doses of lipoic acid. The renal platinum concentration increased following cisplatin administration, which was possibly decreased by chelation with lipoic acid. The data suggest that the graded doses of lipoic acid effectively prevented a decrease in renal antioxidant defense system and prevented an increase in lipid peroxidation, platinum content and plasma creatinine concentrations in a dose-dependent manner.

Effect of cisplatin on the expression of glutathione-S-transferase in the cochlea of the rat.

Glutathione-S-transferase (GST) has been found to conjugate glutathione (GSH) to diverse electrophiles and play a major role in the detoxification of alkylating and platinating agents. However, there is little information regarding the pattern of GST expression in the cochlea. Although cisplatin is ototoxic, its effect on GST in the cochlea is unknown. In the present study we investigated the pattern of GST expression in control and cisplatin-treated cochleas by using the laboratory rat as animal model. Sixteen mature rats were randomly assigned to control or cisplatin groups. After treatment, cochleas were procured and tissues stained by immunohistochemical methods to detect the presence of GST. Optical density measurements were determined to gauge intensity of GST immunostaining. Strong positive GST immunostaining was demonstrated in all cochleas, with the most intense staining found in the spiral ligament and the least in Reissner's membrane. Mean optic density scores were lower for cisplatin-treated cochleas than for control cochleas in all areas analyzed. These findings showed that GST is expressed throughout the rat cochlea, with cisplatin treatment causing its decreased expression.

D-Methionine protects against cisplatin damage to the stria vascularis.

D-Methionine (D-met) protects against cisplatin (CDDP)-induced hearing loss and outer hair cell loss (Campbell et al., 1996). However, D-met's protective effects on the stria vascularis has not been previously investigated. The purpose of this study was to examine, using semi-quantitative analysis, whether D-met also protects the stria vascularis. We removed a basal turn section of the stria vascularis from five groups of five male Wistar rats each: (1) a CDDP-treated control group receiving a 30 min i.p. infusion of 16 mg/kg CDDP, (2) a saline-injected control group receiving an equivalent volume of saline, and (3) three groups injected with either 75, 150, or 300 mg/kg D-methionine (D-met) i.p. 30 min prior to receiving the 16 mg/kg CDDP dosing. Using transmission electron microscopy and light microscopy, we analyzed strial volume (i.e. edema), marginal cell damage classification (bulging and/or compression), and relative optical density (ROD) ratios (i.e. depletion of marginal cell cytoplasmic organelles). All three levels of D-met provided complete protection against marginal cell bulging and/or compression but only partial protection against strial edema. At 300 mg/kg, D-met significantly reduced ROD ratio degradation in the spiral prominence and middle stria vascularis regions. In Reissner's membrane region, values from the D-met pretreated group were not significantly different from either the treated or untreated control groups suggesting only partial protection for that area. Protection of marginal cell cytoplasmic organelles was also noted. In summary, D-met partially or fully protects the stria vascularis from several types of CDDP-induced damage.

Application of antioxidants and other agents to prevent cisplatin ototoxicity.

OBJECTIVE/HYPOTHESIS: To review the recent data from experiments performed in this laboratory to test the hypothesis that cisplatin ototoxicity is related to depletion of glutathione and antioxidant enzymes in the cochlea and that the use of antioxidants or protective agents would protect the cochlea against cisplatin damage and prevent hearing loss. STUDY DESIGN/METHODS: Data were reviewed from experiments performed in this laboratory. Control rats were treated intraperitoneally with cisplatin 16 mg/kg. Experimental rats were given cisplatin in combination with one of the following protective agents: diethyldithiocarbamate, 4-methylthiobenzoic acid, ebselen, or lipoic acid. Animals in each group underwent auditory brainstem response (ABR) threshold testing before and 3 days after treatment. Cochleae were removed after final ABR testing and analyzed for glutathione and activities of the enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and malondialdehyde. RESULTS: Rats in the control group receiving cisplatin were found to have significant ABR threshold shifts. This was accompanied by a reduction of glutathione and the activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase) and an elevation of malondialdehyde. Experimental animals had preservation of ABR thresholds and levels of glutathione, antioxidant enzyme activity, and malondialdehyde that were similar to untreated animals. CONCLUSION: Cisplatin ototoxicity appears to be initiated by fee-radical production, which causes depletion of glutathione and antioxidant enzymes in the cochlea, and lipid peroxidation, manifested by an increase in malondialdehyde. These effects were blocked by each of a series of antioxidant compounds given in combination with cisplatin. A mechanism for cisplatin ototoxicity is elaborated with a proposed plan of chemoprevention using agents with different mechanisms of action. These substances could be used alone or in combination to reduce the severity of cisplatin ototoxicity in patients.

Effect of 4-methylthiobenzoic acid on cisplatin-induced ototoxicity in the rat.

The purpose of this study was to investigate the effectiveness of 4-methylthiobenzoic acid (MTBA) as a protection agent against cisplatin (CDDP)-induced changes in organ of Corti surface structure, compared to electrophysiological changes. Electrophysiological change was assessed using auditory brainstem response (ABR) and morphological changes were assessed using scanning electron microscopy (SEM). Male Wistar rats underwent pre-treatment ABRs in response to clicks, and tone bursts at 2, 4, 8, 16, and 32 kHz. The three groups of rats were injected as follows: (1) MTBA (250 mg/kg, i.p.), (2) CDDP (16 mg/kg, i.p.), (3) CDDP+MTBA (16 mg/kg, i.p. + 250 mg/kg, i.p.). Post-treatment ABRs were performed 3 days after drug administration and rats were sacrificed. Their cochleae were harvested and SEM was used to examine the surface of the organ of Corti, specifically the number of inner hair cells (IHCs) and outer hair cells (OHCs) in the apical, middle and basal turns of the cochlea. Animal weight was measured on the first and final days. There was a good correlation between ABR threshold changes and hair cell loss in the high frequency region of the cochlea (basal turn), while threshold changes in the lower test frequencies (middle turn) appeared to be the result of more subtle changes in the cochlea. MTBA provided effective protection against cisplatin-induced ABR threshold changes at all test frequencies as well as hair cell loss. MTBA also protected against body weight loss.

The effect of combined administration of cadmium and furosemide on auditory function in the rat.

A number of heavy metals have been associated with toxic effects to the peripheral or central auditory system. These include lead, arsenic, mercury, platinum and organic tin compounds. In addition, the ototoxic effects of some metals may be potentiated by other factors. However, the auditory effects of cadmium have not previously been reported. The purpose of the present study was to investigate the potential ototoxic effects of cadmium from an acute dosage, and its potentiation by furosemide. Auditory brainstem response (ABR) thresholds were measured in adult Sprague-Dawley rats. Rats received either cadmium chloride (5 mg/kg, i.p.) followed by saline (4 ml/kg, i.p.). cadmium chloride followed by furosemide (200 mg/kg, i.p.), or furosemide alone. Follow-up ABRs were carried out 7 days post-treatment and threshold changes were compared between each treatment group. No significant threshold change was seen for the cadmium chloride plus saline treated or the furosemide treated animals. However, significant threshold elevations were observed in animals receiving cadmium chloride plus furosemide. In addition, scanning electron microscopy revealed extensive hair cell loss in animals treated with cadmium chloride and furosemide. Although functional auditory changes were not seen after the administration of cadmium alone, the potentiation of threshold changes by furosemide suggests that cadmium may be ototoxic under certain conditions.

Dose dependent protection by lipoic acid against cisplatin-induced ototoxicity in rats: antioxidant defense system.

This study investigated the alterations that occur in auditory brainstem-evoked responses (ABRs) concurrent with changes in cochlear concentrations of glutathione (GSH), lipid peroxidation, and antioxidant enzyme activity in cisplatin-induced ototoxicity and in dose-dependent otoprotection by an antioxidant lipoate. Male Wistar rats were divided into different groups and were treated as follows, with: (1) vehicle (saline) control; (2) cisplatin (16 mg/kg, i.p.); (3) lipoate (100 mg/kg, i.p.) plus saline; (4) cisplatin plus lipoate (25 mg/kg); (5) cisplatin plus lipoate (50 mg/kg), and (6) cisplatin plus lipoate (100 mg/kg). Post-treatment ABRs were evaluated after three days, the rats were sacrificed, and cochleae were harvested and analyzed. The cisplatin-injected rats showed ABR threshold elevations above the pre-treatment thresholds. Rats treated with lipoate plus cisplatin did not show significant elevation of hearing thresholds. Cisplatin administration resulted in a depletion of cochlear GSH concentration (69% of control), whereas, cisplatin-plus-lipoate treatment increased GSH concentration close to control value. Cisplatin-treated rats showed a decrease in cochlear superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) activities (57, 78, 59, and 58% of control, respectively), and an increase in malondialdehyde (MDA) concentration (196% of control). Cochlear SOD, CAT, GSH-Px, and GR activities and MDA concentrations were restored in the rats injected with cisplatin plus graded doses of lipoate than those with cisplatin alone. It is concluded that cisplatin-induced ototoxicity is related to impairment of the cochlear antioxidant defense system, and the dose-dependent otoprotection conferred by an antioxidant lipoate against cisplatin ototoxicity is associated with sparing of the cochlear antioxidant defense system.

Ototoxicity. Amelioration by protective agents.

The findings of studies from this laboratory are summarized to compare the efficacy of four chemoprotective agents against the effects of cisplatin-induced hearing loss and biochemical damage in the rat cochlea. A number of studies have shown that cisplatin is ototoxic, resulting in hearing loss, morphologic damage, and biochemical changes in the cochlea. These studies used Wistar rats, which underwent pre- and posttreatment ABR testing using clicks and tonebursts stimuli at 8, 16, and 32 kHz. Controls received i.p. saline injection. Cisplatin-treated rats were given 16 mg/kg cisplatin i.p. Animals received protective agents in the following dosage: DDTC protected rats received 600 mg/kg subcutaneously an hour after cisplatin. MTBA-protected animals were given 250 mg/kg i.p. 30 minutes before cisplatin. Animals protected with ebselen received 16 mg/kg i.p. an hour before cisplatin. One hundred mg/kg of alpha-lipoic acid was injected i.p. 30 minutes before cisplatin. Rats were sacrificed three days after treatment and the cochleae were harvested and frozen in liquid nitrogen and stored at -80 degrees C until analysis of glutathione (GSH), the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and malondialdehyde was performed. Cisplatin-treated rats were found to have ABR threshold shifts of 27-40 dB, and rats treated with chemoprotective agents plus cisplatin all had ABR thresholds shifts of less than 10 dB. Significant depletion of glutathione and decrease of the activities of the antioxidant enzymes were observed in cisplatin-treated rats. These changes were accompanied by a marked elevation of malondialdehyde. These changes were almost completely prevented by the use of the chemoprotective agents. These findings suggest that cisplatin ototoxicity is related to lipid peroxidation and that the use of protective agents prevents hearing loss and lipid peroxidation by sparing the antioxidant defense system in the cochlea.

Experimental model of cisplatin ototoxicity in chinchillas.

Cisplatin is an important antineoplastic agent. Its ototoxicity has been well defined, both in human and animal studies. However, animal models of systemic cisplatin administration have been complicated by multiple toxic effects. We studied cisplatin ototoxicity in an animal model involving topical application of cisplatin to the round-window membrane. Adult chinchillas were anesthetized with ketamine and pentobarbital, and auditory function was tested with the use of auditory brain-stem responses to various stimuli (clicks and 8-and 16-kHz tone bursts). Each animal was used as its own control. The middle-ear cavity was exposed through the bulla. In the experimental ear, a 25-microl solution of 0.25 mg cisplatin/1.0 ml normal saline solution was applied to the round-window membrane. In the control ear, 25 microl normal saline solution was applied to the round-window membrane. Follow-up auditory brain-stem response testing was conducted 7 days after treatment. A significant increase in threshold in the experimental ears was seen on comparison with the control ears. This finding suggests that application of cisplatin to the round-window membrane is a useful animal model in which to study cisplatin ototoxicity.

A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis.

Cisplatin (CDDP) is a very effective chemotherapeutic agent but is highly ototoxic. Most studies have focused on the effects of CDDP on the outer hair cells. The purpose of this study was to examine changes in the stria vascularis in cisplatin treated male Wistar rats and to provide semiquantitative analysis of the results. We removed a section of the stria vascularis from the basal turn of five control and five CDDP (16 mg/kg) treated rats. Using transmission electron microscopy (TEM) we analyzed: (1) changes to the strial tissue as a whole; and (2) intracellular changes in the marginal cells. We also subjected the samples to semiquantitative analysis using the MCID, focusing on three aspects of strial profile abnormalities; the number of abnormal marginal cells in CDDP treated tissue, intracellular strial edema and densitometry. Controls appeared normal, but many pathologic changes were apparent in the experimental group. Results from the semiquantitative analysis indicate cisplatin has a deleterious effect on the stria vascularis including strial edema; bulging, rupture and/or compression of the marginal cells and depletion of the cytoplasmic organelles.

Interaction of methylprednisolone and transient asphyxia on the inner ear of the adrenalectomized rat.

Methylprednisolone has been shown clinically to have beneficial effects on certain types of hearing loss. In the current study, compound action potential (CAP) thresholds, endocochlear potentials (EPs), and potassium concentration (CK+) values in the endolymph were determined under conditions of transient asphyxia (45 seconds) and methylprednisolone treatment (24 hours) in bilateral adrenalectomized rats. Treatment with methylprednisolone significantly reduced the effect of transient asphyxia on CAP thresholds as compared with nontreated animals. Methylprednisolone did not alter the dramatic short-term reduction in the EPs produced by anoxia. Potassium concentrations in treated adrenalectomized rats were significantly lower before transient asphyxia than in nontreated adrenalectomized rats. In the nontreated rats, transient asphyxia induced a reduction in CK+ levels that was not seen in the methylprednisolone-treated animals. The data support the clinical application of methylprednisolone for certain forms of hearing loss and for potassium imbalance in the endolymph.