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    • Otolaryngology– Head and Neck Surgery NOVEMBER 2003 VOLUME 129 NUMBER 5 ORIGINAL ARTICLES Effects of resveratrol on acoustic trauma MICHAEL SEIDMAN, MD, SEILESH BABU, MD, WENXUE TANG, MD, EMAD NAEM, MD, and WAYNE S. QUIRK, PHD, Detroit, Michigan, and Ellensburg, Washington OBJECTIVE: The purpose of the study is to test the points: immediate, at 3 days, 7 days, and 4 weeks ability of resveratrol to protect the auditory system after noise exposure. from reactive oxygen species (ROS)-mediated RESULTS: Results demonstrate that the resveratrol noise damage. Oxidative stress is mediated by group showed reduced threshold shifts compared ROS, which are known to cause cellular and mo- with the control group after noise exposure. These lecular damage. Interfering with this process, using shifts were significantly different between groups at ROS inhibitors/scavengers such as antioxidants has 6 and 9 kHz (P < 0.05), corresponding to the region shown promise in protecting specific systems from most represented by the frequency of the traumatic oxidative damage. Among the antioxidants receiv- noise. ing recent attention is resveratrol, an active com- CONCLUSION/SIGNIFICANCE: Initial studies in our ponent in red wine. laboratory as well as other investigators have STUDY DESIGN AND SETTING: Ten Fischer rats were shown the importance of specific antioxidant ther- used for this study. The experimental group (n 5) apy in the prevention of ischemic, noise, and age received 7 weeks of resveratrol treatment (430/ g/ related hearing loss. The current study demon- kg/day), by gavage, and the control group (n 5) strates a protective effect of resveratrol on noise- received normal saline solution by gavage. Base- induced hearing loss. (Otolaryngol Head Neck line auditory brainstem responses (3, 6, 9, 12 and 18 Surg 2003;129:463-70.) kHz) were determined for both groups. After 21 days, animals were exposed to noise (105 dB, 4500 N oise-induced hearing loss (NIHL) is a problem to 9000 Hz for 24 hours). Postnoise auditory brain- of epidemic proportions. The National Institute of stem responses were assessed at 4 recovery time Deafness and Other Communication Disorders re- cently estimated that 10 million Americans have From the Department of Otolaryngology (Drs Seidman, irreversible acoustic damage and more than 30 Babu, Tang, and Naem), Henry Ford Health System, De- million people are exposed to dangerous levels of troit, Michigan, and Central Washington University (Dr noise on a daily basis. Additionally, NIHL is a Quirk), Ellensburg, Washington. Presented at American Academy of Otolaryngology Head and leading work-related disease and injury. Accord- Neck Surgery Meeting in Denver, Colorado, September ing to the World Health Organization, it is esti- 2001. mated that the cost of NIHL in developed coun- Reprint requests: Michael Seidman, MD, Department of Oto- tries ranges from 0.2% to 2% of the gross national laryngology, Henry Ford Health System, One Ford Place, product.1 Detroit, MI 48202; e-mail, mseidma1@hfhs.com. There are many unresolved issues concerning Copyright © 2003 by the American Academy of Otolaryn- gology–Head and Neck Surgery Foundation, Inc. NIHL. For instance, uncertainties exist over sus- 0194-5998/2003/$30.00 0 ceptibility, prevention, and treatment of NIHL. A doi:10.1016/S0194-5998(03)01586-9 variety of experimental strategies have been used 463
    • Otolaryngology– Head and Neck Surgery 464 SEIDMAN et al November 2003 to affect acoustic damage. Some interventions that mg/kg and 15 mg/kg, respectively) given subcu- reduce acoustic damage include lowering the body taneously. temperature, stimulating the efferent olivocochlear Auditory stimuli were generated using a D/A bundle, treating with various pharmacological converter (Model DA3-2; Tucker-Davis Technol- compounds or endogenous factors, and increasing ogies (TDT), Gainesville, FL, USA) with a sam- oxygenation with carbogen or oxygen.2,3 Studies pling frequency of 100 kHz. The output of the D/A from our laboratory and others have achieved sim- converter was connected to a programmable at- ilar partial protection from noise damage by inter- tenuator (Model PA4; TDT), a weighted summer fering with the activity of reactive oxygen species. (Model SM3; TDT), a headphone buffer (Model Resveratrol, found mainly in the skin of grapes, is HB6; TDT), and an earphone (Model DT-48; noted for its antioxidant and anti-inflammatory Beyer Dynamic, Heilbronn, Germany), which was properties, as well as its potential to prevent can- placed within 1 to 2 mm from the tympanic mem- cer and heart disease. Epidemiological evidence brane of the animal. The stimuli consisted of tone has shown that moderate consumption of red wine bursts with a rise/fall time of 1 ms, a duration of is inversely correlated with the incidence of de- 15 ms, and a period of 100 ms. A series of stimuli mentia and ischemic heart disease.4 Direct neuro- were produced at 3, 6, 9, 12 and 18 kHz test protective effects of resveratrol against oxidative frequencies with intensities ranging from 5 to 100 stress have been demonstrated in PC12 cells.5 dB SPL in 5 dB increments. The system was Resveratrol stimulates a key enzyme in the brain calibrated at the tympanic membrane using a known as Map-kinase, which is involved in nerve probe microphone (Model ER-7C; Etymotic Re- regeneration and neural protection from the dam- search, Elk Grove Village, IL), which was con- age caused by systemic injection of the excito- nected to an A/D converter (Model AD2, TDT) toxin kainic acid.6 Our study will assess the pos- and a computer. Automated calibrating routines sible protective effects of resveratrol on noise were used for online calibration. damage by evaluating noise-induced temporary Auditory brainstem responses were collected threshold shifts (TTS), permanent threshold shifts using subcutaneous electrodes (Model E2; Grass (PTS), and inner ear histologic changes. Instruments, Quincy, MA, USA) placed at the vertex and under both pinnae of each animal. This MATERIAL AND METHODS output was channeled into a biologic amplifier Subjects (Model P5 Series; Grass Instruments) with a gain of 100,000X. The response was filtered between Ten healthy male Fischer rats (130 to 175 gm) 0.3 and 3.0 kHz, and then the output was sent to an were used to study noise-induced TTS and PTS as A/D converter (Model AD2; TDT). Custom-de- determined by auditory brainstem response (ABR) signed software allowed these responses to be measurements. These animals were randomly as- displayed with a sampling rate of 50 kHz in real signed to either the resveratrol treatment group (n time on a computer monitor. For each recording, a 5) or the control group (n 5). The use of 20-ms neural response was averaged 1,024 times. experimental animals for this study was approved For each of the 5 test frequencies, auditory thresh- by the Care for Experimental Animals Committee olds were determined by identifying the smallest at Henry Ford Health System. Additionally, all intensity (in dB SPL) at which ABR waveforms procedures were conducted in strict compliance became consistently evident at 1 mV responses. with the National Institutes of Health guidelines This was determined objectively by the computer for experimental animal subjects. and confirmed by direct observation of the wave- form. Auditory Brainstem Response (ABR) After baseline ABR testing, treatment group Baseline ABR measurements were obtained animals (n 5) received 3 weeks of trans-resvera- from these 10 healthy male Fischer rats at initial trol (430/ug/kg/day) treatment by gavage feeding. entry into the study. Animals were anesthetized Trans-resveratrol was purchased from Sigma (St with a mixture of ketamine and xylazine (100 Louis, MO) and was dissolved in 100% ethanol
    • Otolaryngology– Head and Neck Surgery Volume 129 Number 5 SEIDMAN et al 465 Fig 1. Baseline auditory brainstem response thresholds. Mean auditory threshold levels of treatment group (dark line) and control group (light line) at 5 test frequencies. Measurements were obtained before treatment with resveratrol. (Bars represent Standard Error of the mean.) (10 mg/ml), stocked at –20°C, and diluted with aortic perfusion. The bullae were rapidly removed 0.9% NaCl to final ethanol concentration of 2.5% and the cochleae were perfused in vitro with a vol/vol as necessary. The control group (n 5) 2.5% glutaraldehyde solution in veronal acetate was gavaged with an equivalent volume of 0.9% buffer (pH 7.4) through the oval and round NaCl. The animals were then placed in an acous- windows. The tissues were fixed in the same so- tically insulated noise booth (Industrial Acoustics, lution at 4°oC for 48 hours. The cochleae were New York, NY) and exposed to 105 dB SPL at perfused with 1% osmium tetroxide in veronal 4500 to 9000 Hz narrow band noise for 24 con- acetate buffer (pH 7.4) through the oval and tinuous hours. After the completion of the noise round windows for 30 minutes. The cochleae were exposure, experimental group animals received dehydrated using ethanol of ascending strength another 4 weeks of resveratrol (430/ug/kg/day) by from 30% through 50% to 70% and decalcified gavage, and the control group was gavaged with overnight in 0.35 M ethylenediaminetetracetic an equivalent volume of 0.9% NaCl. Although acid (EDTA) in veronal acetate buffer (pH 7.4). caging was designed to be acoustically transpar- The organ of Corti was then dissected in 70% ent, calibration measurements throughout the cage ethanol. Each cochlear turn was mounted with revealed a variance of 2 to 3 dB from the front of 99% glycerol and examined under an optical mi- the cage to the back. To determine temporary croscope. The hair cells were counted at 400 threshold shifts (TTS) and permanent threshold using a differential interference contrast micro- shifts (PTS), ABR measurements were recorded at various times: immediately, 3 days, 7 days, and 4 scope (Carl Zeiss GFL, Goettingen, Germany). weeks after the completion of the noise exposure. These data were then used to generate a cytoco- chleogram as a frequency-position map based on Cochlear Histology the following mathematical derivation: f (kHz) At the end of the 7-week study period, 8 of the 3.109 * (10 (100-d)*0.0142) - 0.7719), where d is the animals involved in the auditory sensitivity studies percent distance from the cochlear base.7 were sacrificed; 4 from the resveratrol treatment Outer and inner hair cell loss was evaluated group and 4 from the control group. One animal in for each animal as a percent hair cell loss and each group had expired before the cochleae was plotted against basilar membrane distance (cal- able to be harvested. The cochleae were perfused culated from the cochlear apex). The basilar in vivo with a 2.5% glutaraldehyde solution in membrane length was also matched for frequen- veronal acetate buffer (pH 7.4) via transcardiac cy-position. The pattern of hair cell loss was
    • Otolaryngology– Head and Neck Surgery 466 SEIDMAN et al November 2003 Fig 2. Auditory treshold shifts. Mean auditory shifts measured immediately (A), 3 days (B), 7 days (D), and 4 weeks (D) after high-level noise exposure. Dark lines represent resveratrol treatment group and light lines represent control group at 5 test frequencies (3, 6, 9, 12, and 18 kHz). (Standard Error bars are plotted for each condition.) compared between the treatment and control and 4 weeks after noise exposure (SigmaStat Soft- groups. ware). Significant differences in audiologic data between the 2 groups were assessed using Stu- RESULTS dent’s t test. Auditory Brainstem Response (ABR) Also reductions were noted in threshold shifts at ABR thresholds were recorded at 5 test frequen- 12 kHz in the immediate and 7 day time points. cies (3, 6, 9, 12, and 18 kHz) and 5 time points (baseline, 0, 3, 7 days, and 4 weeks after noise Cochlear Histology exposure) as described previously. ABR measure- Figure 3 displays surface preparations of the ments obtained before resveratrol treatment indi- organ of Corti from a control ear and a resveratrol cated that there were no significant differences in treated ear from representative animals. All of the mean auditory thresholds between the treatment selected animals showed outer hair cell loss, but and control groups at baseline (Fig 1). Subsequent the loss was significantly less in the resveratrol mean auditory threshold shifts from baseline were treated group. Figure 4 depicts a mean cytoco- also graphed (Fig 2). Statistically significant re- chleogram for both the control and resveratrol ductions in auditory threshold shifts were noted treatment groups. Outer hair cell (OHC) loss was when comparing the resveratrol treatment group to determined over 0.24 mm intervals along the co- the control group at 2 test frequencies (6 kHz and chlea from apex to base. The mean basilar mem- 9 kHz) at all 4 times: immediately, 3 days, 7 days, brane length in the control group was 8.9 mm. The
    • Otolaryngology– Head and Neck Surgery Volume 129 Number 5 SEIDMAN et al 467 Fig 3. Cochlear histology. Photomicrographs ( 40) of surface mount preparation of the organ of Corti from a control ear (A) and resveratrol-treated ear (B). (Three outer hair cell rows are labeled 1, 2, and 3; I, single hair cell row; arrowhead points to outer hair cells loss.) mean basilar membrane length in the resveratrol inner hair cells was not detected in either group of treatment group was 8.97 mm. The mean OHC animals. loss was 1.3% in the control group. OHC loss in the resveratrol treatment group was 0.48%. In the DISCUSSION control group, peak outer hair cell loss was 5.2%, The current study shows that treatment with corresponding to 7 to 9 kHz. In the resveratrol resveratrol prevents significant noise-induced group, peak outer hair cell loss was 2.7%, corre- hearing loss based on auditory brainstem response sponding to 7 to 8 kHz. A significant change of the testing and histologic examination. Statistically
    • Otolaryngology– Head and Neck Surgery 468 SEIDMAN et al November 2003 Fig 4. Cochlear histology. Mean cytocochleograms of control (dark line) and rseveratrol-treated group (light line) show percentage of hair cell loss as a function of frequency position on basilar lamina. significant reductions in auditory threshold shifts Hypoperfusion and ischemia to the cochlea is a were noted when comparing the resveratrol treat- possible mechanism of damage associated with ment group to the control group at 2 test frequen- noise-induced hearing loss. Ischemia, which is cies (6 and 9 kHz) at all 4 time points: immedi- known to cause injury to the cochlea, results in ately, 3 days, 7 days, and 4 weeks after noise oxidative stress that stimulates the generation and exposure. The traumatic noise exposure consisted release of reactive oxygen species (ROS).8 These of frequencies between 4500 and 9000 Hz, thus ROS affect energy production and ultimately re- accounting for the threshold shift at 6 and 9 KHz duce outer hair cell function. This damage is evi- and the protective effects of resveratrol at these dent in the auditory system in the form of thresh- frequencies as well. Additionally, a greater loss of old shifts. Many studies have shown signs of hair cells was found in the control group compared vascular insufficiency following noise expo- with the resveratrol treated group. This demon- sure.9-11 Our in vivo studies have shown hypoper- strates a protective effect of resveratrol on the fusion and ischemia in the cochlear microcircula- cochlea. Although unlikely this effect may have tion during noise exposure.12 Reductions in been influenced by the use of ethanol as the dis- cochlear blood flow, such as occurs with noise solving agent for resveratrol. Ongoing studies are exposure, have long been known to decrease au- investigating the role of ethanol causing a protec- ditory sensitivity.13 Indeed, recent evidence sug- tive role. gests an increase in ROS following noise expo-
    • Otolaryngology– Head and Neck Surgery Volume 129 Number 5 SEIDMAN et al 469 sure.14 Several studies from our laboratories have tection; and neuroprotection.24-28 Bertelli et al29 shown ROS scavenger treatment in the auditory investigated the absorption, the concentration in system reduces these threshold shifts following different organs, and the excretion of natural trans- experimentally induced cochlear ischemia.15,16 and cis-Resveratrol after red wine oral administra- ROS are also implicated in other ototoxic insults tion to rats. Their results show that prolonged such as cisplatin, trimethyltin, and aminoglyco- administration of red wine in the diet could lead to sides.5,9,17,18 Studies have also demonstrated that an increased resveratrol concentration in different a reduction of ROS attenuates noise-induced hear- tissues even though the amount of resveratrol in ing loss.19,20 In summary, noise results in isch- these different tissues was lower than that required emia, a condition known to affect auditory thresh- for pharmacological activity. This may explain its olds. This ischemia results in the generation of beneficial role against coronary heart disease. ROS and the damaging effects of these radicals Oxidative stress in the central nervous system can be attenuated with scavengers and inhibitors may cause oxidation of lipoprotein particles. The in the auditory system. oxidized lipoproteins may damage cellular and Resveratrol is found in over 70 fruits and plants, subcellular membranes, leading to tissue injury many of which are edible, such as mulberries, and cell death. Draczynska-Lusiak et al30 have peanuts, and grapes. It is thought that this chem- shown that antioxidants, such as vitamins E or C, ical is produced in response to environmental or resveratrol, protect neuronal cell damage from stress or attack by pathogens including mold. oxidative stress in vitro. Results indicated that Grapes contain particularly high concentrations of oxidized lipoproteins may serve as an oxidative resveratrol in the skin. Trans-resveratrol was first stressor, which may initiate the neuronal cell death detected in grapevines in 1976 by Langcake and leading to the manifestation of Alzheimer disease. Pryce,21 who found that the compound was syn- Zini et al31 studied the possible effects of resvera- thesized by leaf tissues in response to fungal in- trol on the mitochondrial respiratory chain in rat fection or exposure to ultraviolet light.5 Resvera- brains. Resveratrol was found to decrease complex trol was brought to the public attention when its III activity in rat brain by competition with coen- presence in wine was reported in 1992 by Siemann zyme Q. This property is especially interesting as and Creasy.22 The authors suggested that this this complex is the site where reactive oxygen compound might be the biologically active ingre- substances (ROS) are generated. By decreasing dient of red wine.23 The consumption of red wine the activity of complex III, resveratrol not only is becoming increasingly popular due to the in- opposes the production of ROS but also scavenges trigue created by the French Paradox. Despite the them.31 Virgili and Contestabile14 report that high fat diet and smoking tendencies of the pop- chronic administration of resveratrol to young ulation in Southern France, there is an astonishing adult rats, significantly protects from the damage 42% lower incidence of heart disease than that caused by systemic injection of the excitotoxin found in Americans.4 The effect known as the kainic acid in the olfactory cortex and the hip- French Paradox has been attributed to the con- pocampus. sumption of red wine. World Health Organization Several studies have demonstrated that antioxi- data indicate that resveratrol may be one of the dants can attenuate hearing loss in various condi- active ingredients in the wine that reduces the risk tions, such as noise-induced hearing loss, ototox- of coronary heart disease by up to 40% in red wine icity, ischemia, and presbycusis.2,16,32-33 drinkers.1 Resveratrol has many important biologic activ- CONCLUSION ities including: inhibition of lipid peroxidation; Considering the importance of the biological chelation of copper; free-radical scavenging; alter- activities of resveratrol, along with previous stud- ation of eicosanoid synthesis; inhibition of platelet ies from our laboratory showing the protective aggregation; anti-inflammatory activity; vasore- effects of antioxidants and dietary restriction on laxing activity; modulation of lipid metabolism; noise-induced and age-induced hearing loss, the anticancer activity; estrogenic activity; cardiopro- current study demonstrates similar effects with
    • Otolaryngology– Head and Neck Surgery 470 SEIDMAN et al November 2003 resveratrol. Thus, resveratrol appears to protect 18. Hester T, Jones R, Clerici W. Protection against aminogly- the cochlea from acoustic trauma and ongoing coside otic drop-induced ototoxicity by a spin trap. I. acute effects. Otolaryngol Head Neck Surg 1998;119:581-7. studies are being done to evaluate the efficacy of 19. Kopke R, Liu W, Gabazadeh R, et al. Use of organotypic resveratrol on age-related hearing loss. cultures of Corti’s organ to study the protective effects of antioxidant molecules on cisplatin-induced damage of REFERENCES auditory hair cells. Am J Otol 1997;18:559-71. 1. United States Department of Health and Human Servic- 20. Seidman M, Quirk W, Nuttall A, et al. The protective es–Public Health Services. 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