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ORIGINAL ARTICLES

Effects of resveratrol on acoustic traumaMICHAEL 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 theability of resveratrol to protect the auditory systemfrom reactive oxygen species (ROS)-mediatednoise damage. Oxidative stress is mediated byROS, which are known to cause cellular and mo-lecular damage. Interfering with this process, usingROS inhibitors/scavengers such as antioxidants hasshown promise in protecting specific systems fromoxidative damage. Among the antioxidants receiv-ing recent attention is resveratrol, an active com-ponent in red wine.STUDY DESIGN AND SETTING: Ten Fischer rats wereused for this study. The experimental group (n � 5)received 7 weeks of resveratrol treatment (430/�g/kg/day), by gavage, and the control group (n � 5)received normal saline solution by gavage. Base-line auditory brainstem responses (3, 6, 9, 12 and 18kHz) were determined for both groups. After 21days, animals were exposed to noise (105 dB, 4500to 9000 Hz for 24 hours). Postnoise auditory brain-stem responses were assessed at 4 recovery time

points: immediate, at 3 days, 7 days, and 4 weeksafter noise exposure.RESULTS: Results demonstrate that the resveratrolgroup showed reduced threshold shifts comparedwith the control group after noise exposure. Theseshifts were significantly different between groups at6 and 9 kHz (P < 0.05), corresponding to the regionmost represented by the frequency of the traumaticnoise.CONCLUSION/SIGNIFICANCE: Initial studies in ourlaboratory as well as other investigators haveshown the importance of specific antioxidant ther-apy in the prevention of ischemic, noise, and agerelated hearing loss. The current study demon-strates a protective effect of resveratrol on noise-induced hearing loss. (Otolaryngol Head NeckSurg 2003;129:463-70.)

Noise-induced hearing loss (NIHL) is a problemof epidemic proportions. The National Institute ofDeafness and Other Communication Disorders re-cently estimated that 10 million Americans haveirreversible acoustic damage and more than 30million people are exposed to dangerous levels ofnoise on a daily basis. Additionally, NIHL is aleading work-related disease and injury. Accord-ing to the World Health Organization, it is esti-mated that the cost of NIHL in developed coun-tries ranges from 0.2% to 2% of the gross nationalproduct.1

There are many unresolved issues concerningNIHL. For instance, uncertainties exist over sus-ceptibility, prevention, and treatment of NIHL. Avariety of experimental strategies have been used

From the Department of Otolaryngology (Drs Seidman,Babu, Tang, and Naem), Henry Ford Health System, De-troit, Michigan, and Central Washington University (DrQuirk), Ellensburg, Washington.

Presented at American Academy of Otolaryngology Head andNeck Surgery Meeting in Denver, Colorado, September2001.

Reprint requests: Michael Seidman, MD, Department of Oto-laryngology, Henry Ford Health System, One Ford Place,Detroit, MI 48202; e-mail, [email protected].

Copyright © 2003 by the American Academy of Otolaryn-gology–Head and Neck Surgery Foundation, Inc.

0194-5998/2003/$30.00 � 0doi:10.1016/S0194-5998(03)01586-9

Otolaryngology–Head and Neck Surgery

NOVEMBER 2003 VOLUME 129 NUMBER 5

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to affect acoustic damage. Some interventions thatreduce acoustic damage include lowering the bodytemperature, stimulating the efferent olivocochlearbundle, treating with various pharmacologicalcompounds or endogenous factors, and increasingoxygenation with carbogen or oxygen.2,3 Studiesfrom our laboratory and others have achieved sim-ilar partial protection from noise damage by inter-fering with the activity of reactive oxygen species.Resveratrol, found mainly in the skin of grapes, isnoted for its antioxidant and anti-inflammatoryproperties, as well as its potential to prevent can-cer and heart disease. Epidemiological evidencehas shown that moderate consumption of red wineis inversely correlated with the incidence of de-mentia and ischemic heart disease.4 Direct neuro-protective effects of resveratrol against oxidativestress have been demonstrated in PC12 cells.5

Resveratrol stimulates a key enzyme in the brainknown as Map-kinase, which is involved in nerveregeneration and neural protection from the dam-age caused by systemic injection of the excito-toxin kainic acid.6 Our study will assess the pos-sible protective effects of resveratrol on noisedamage by evaluating noise-induced temporarythreshold shifts (TTS), permanent threshold shifts(PTS), and inner ear histologic changes.

MATERIAL AND METHODSSubjects

Ten healthy male Fischer rats (130 to 175 gm)were used to study noise-induced TTS and PTS asdetermined by auditory brainstem response (ABR)measurements. These animals were randomly as-signed to either the resveratrol treatment group (n� 5) or the control group (n � 5). The use ofexperimental animals for this study was approvedby the Care for Experimental Animals Committeeat Henry Ford Health System. Additionally, allprocedures were conducted in strict compliancewith the National Institutes of Health guidelinesfor experimental animal subjects.

Auditory Brainstem Response (ABR)Baseline ABR measurements were obtained

from these 10 healthy male Fischer rats at initialentry into the study. Animals were anesthetizedwith a mixture of ketamine and xylazine (100

mg/kg and 15 mg/kg, respectively) given subcu-taneously.

Auditory stimuli were generated using a D/Aconverter (Model DA3-2; Tucker-Davis Technol-ogies (TDT), Gainesville, FL, USA) with a sam-pling frequency of 100 kHz. The output of the D/Aconverter was connected to a programmable at-tenuator (Model PA4; TDT), a weighted summer(Model SM3; TDT), a headphone buffer (ModelHB6; TDT), and an earphone (Model DT-48;Beyer Dynamic, Heilbronn, Germany), which wasplaced within 1 to 2 mm from the tympanic mem-brane of the animal. The stimuli consisted of tonebursts with a rise/fall time of 1 ms, a duration of15 ms, and a period of 100 ms. A series of stimuliwere produced at 3, 6, 9, 12 and 18 kHz testfrequencies with intensities ranging from 5 to 100dB SPL in 5 dB increments. The system wascalibrated at the tympanic membrane using aprobe microphone (Model ER-7C; Etymotic Re-search, Elk Grove Village, IL), which was con-nected to an A/D converter (Model AD2, TDT)and a computer. Automated calibrating routineswere used for online calibration.

Auditory brainstem responses were collectedusing subcutaneous electrodes (Model E2; GrassInstruments, Quincy, MA, USA) placed at thevertex and under both pinnae of each animal. Thisoutput was channeled into a biologic amplifier(Model P5 Series; Grass Instruments) with a gainof 100,000X. The response was filtered between0.3 and 3.0 kHz, and then the output was sent to anA/D converter (Model AD2; TDT). Custom-de-signed software allowed these responses to bedisplayed with a sampling rate of 50 kHz in realtime on a computer monitor. For each recording, a20-ms neural response was averaged 1,024 times.For each of the 5 test frequencies, auditory thresh-olds were determined by identifying the smallestintensity (in dB SPL) at which ABR waveformsbecame consistently evident at 1 mV responses.This was determined objectively by the computerand confirmed by direct observation of the wave-form.

After baseline ABR testing, treatment groupanimals (n � 5) received 3 weeks of trans-resvera-trol (430/ug/kg/day) treatment by gavage feeding.Trans-resveratrol was purchased from Sigma (StLouis, MO) and was dissolved in 100% ethanol


464 SEIDMAN et al November 2003

(10 mg/ml), stocked at �–20°C, and diluted with0.9% NaCl to final ethanol concentration of 2.5%vol/vol as necessary. The control group (n � 5)was gavaged with an equivalent volume of 0.9%NaCl. The animals were then placed in an acous-tically insulated noise booth (Industrial Acoustics,New York, NY) and exposed to 105 dB SPL at4500 to 9000 Hz narrow band noise for 24 con-tinuous hours. After the completion of the noiseexposure, experimental group animals receivedanother 4 weeks of resveratrol (430/ug/kg/day) bygavage, and the control group was gavaged withan equivalent volume of 0.9% NaCl. Althoughcaging was designed to be acoustically transpar-ent, calibration measurements throughout the cagerevealed a variance of 2 to 3 dB from the front ofthe cage to the back. To determine temporarythreshold shifts (TTS) and permanent thresholdshifts (PTS), ABR measurements were recorded atvarious times: immediately, 3 days, 7 days, and 4weeks after the completion of the noise exposure.

Cochlear HistologyAt the end of the 7-week study period, 8 of the

animals involved in the auditory sensitivity studieswere sacrificed; 4 from the resveratrol treatmentgroup and 4 from the control group. One animal ineach group had expired before the cochleae wasable to be harvested. The cochleae were perfusedin vivo with a 2.5% glutaraldehyde solution inveronal acetate buffer (pH � 7.4) via transcardiac

aortic perfusion. The bullae were rapidly removedand the cochleae were perfused in vitro with a2.5% glutaraldehyde solution in veronal acetatebuffer (pH � 7.4) through the oval and roundwindows. The tissues were fixed in the same so-lution at 4°oC for 48 hours. The cochleae wereperfused with 1% osmium tetroxide in veronalacetate buffer (pH � 7.4) through the oval andround windows for 30 minutes. The cochleae weredehydrated using ethanol of ascending strengthfrom 30% through 50% to 70% and decalcifiedovernight in 0.35 M ethylenediaminetetraceticacid (EDTA) in veronal acetate buffer (pH 7.4).The organ of Corti was then dissected in 70%ethanol. Each cochlear turn was mounted with99% glycerol and examined under an optical mi-croscope. The hair cells were counted at �400using a differential interference contrast micro-scope (Carl Zeiss GFL, Goettingen, Germany).These data were then used to generate a cytoco-chleogram as a frequency-position map based onthe following mathematical derivation: f (kHz) �3.109 * (10 (100-d)*0.0142) - 0.7719), where d is thepercent distance from the cochlear base.7

Outer and inner hair cell loss was evaluatedfor each animal as a percent hair cell loss andplotted against basilar membrane distance (cal-culated from the cochlear apex). The basilarmembrane length was also matched for frequen-cy-position. The pattern of hair cell loss was

Fig 1. Baseline auditory brainstem response thresholds. Mean auditory threshold levels of treatment group (dark line) andcontrol group (light line) at 5 test frequencies. Measurements were obtained before treatment with resveratrol. (Barsrepresent Standard Error of the mean.)

Otolaryngology–Head and Neck SurgeryVolume 129 Number 5 SEIDMAN et al 465

compared between the treatment and controlgroups.

RESULTSAuditory Brainstem Response (ABR)

ABR thresholds were recorded at 5 test frequen-cies (3, 6, 9, 12, and 18 kHz) and 5 time points(baseline, 0, 3, 7 days, and 4 weeks after noiseexposure) as described previously. ABR measure-ments obtained before resveratrol treatment indi-cated that there were no significant differences inmean auditory thresholds between the treatmentand control groups at baseline (Fig 1). Subsequentmean auditory threshold shifts from baseline werealso graphed (Fig 2). Statistically significant re-ductions in auditory threshold shifts were notedwhen comparing the resveratrol treatment group tothe control group at 2 test frequencies (6 kHz and9 kHz) at all 4 times: immediately, 3 days, 7 days,

and 4 weeks after noise exposure (SigmaStat Soft-ware). Significant differences in audiologic databetween the 2 groups were assessed using Stu-dent’s t test.

Also reductions were noted in threshold shifts at12 kHz in the immediate and 7 day time points.

Cochlear HistologyFigure 3 displays surface preparations of the

organ of Corti from a control ear and a resveratroltreated ear from representative animals. All of theselected animals showed outer hair cell loss, butthe loss was significantly less in the resveratroltreated group. Figure 4 depicts a mean cytoco-chleogram for both the control and resveratroltreatment groups. Outer hair cell (OHC) loss wasdetermined over 0.24 mm intervals along the co-chlea from apex to base. The mean basilar mem-brane length in the control group was 8.9 mm. The

Fig 2. Auditory treshold shifts. Mean auditory shifts measured immediately (A), 3 days (B), 7 days (D), and 4 weeks (D) afterhigh-level noise exposure. Dark lines represent resveratrol treatment group and light lines represent control group at 5 testfrequencies (3, 6, 9, 12, and 18 kHz). (Standard Error bars are plotted for each condition.)



mean basilar membrane length in the resveratroltreatment group was 8.97 mm. The mean OHCloss was 1.3% in the control group. OHC loss inthe resveratrol treatment group was 0.48%. In thecontrol group, peak outer hair cell loss was 5.2%,corresponding to 7 to 9 kHz. In the resveratrolgroup, peak outer hair cell loss was 2.7%, corre-sponding to 7 to 8 kHz. A significant change of the

inner hair cells was not detected in either group ofanimals.

DISCUSSIONThe current study shows that treatment with

resveratrol prevents significant noise-inducedhearing loss based on auditory brainstem responsetesting and histologic examination. Statistically

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; arrowheadpoints to outer hair cells loss.)


significant reductions in auditory threshold shiftswere noted when comparing the resveratrol treat-ment group to the control group at 2 test frequen-cies (6 and 9 kHz) at all 4 time points: immedi-ately, 3 days, 7 days, and 4 weeks after noiseexposure. The traumatic noise exposure consistedof frequencies between 4500 and 9000 Hz, thusaccounting for the threshold shift at 6 and 9 KHzand the protective effects of resveratrol at thesefrequencies as well. Additionally, a greater loss ofhair cells was found in the control group comparedwith the resveratrol treated group. This demon-strates a protective effect of resveratrol on thecochlea. Although unlikely this effect may havebeen influenced by the use of ethanol as the dis-solving agent for resveratrol. Ongoing studies areinvestigating the role of ethanol causing a protec-tive role.

Hypoperfusion and ischemia to the cochlea is apossible mechanism of damage associated withnoise-induced hearing loss. Ischemia, which isknown to cause injury to the cochlea, results inoxidative stress that stimulates the generation andrelease of reactive oxygen species (ROS).8 TheseROS affect energy production and ultimately re-duce outer hair cell function. This damage is evi-dent in the auditory system in the form of thresh-old shifts. Many studies have shown signs ofvascular insufficiency following noise expo-sure.9-11 Our in vivo studies have shown hypoper-fusion and ischemia in the cochlear microcircula-tion during noise exposure.12 Reductions incochlear blood flow, such as occurs with noiseexposure, have long been known to decrease au-ditory sensitivity.13 Indeed, recent evidence sug-gests an increase in ROS following noise expo-

Fig 4. Cochlear histology. Mean cytocochleograms of control (dark line) and rseveratrol-treated group (light line) showpercentage of hair cell loss as a function of frequency position on basilar lamina.



sure.14 Several studies from our laboratories haveshown ROS scavenger treatment in the auditorysystem reduces these threshold shifts followingexperimentally induced cochlear ischemia.15,16

ROS are also implicated in other ototoxic insultssuch as cisplatin, trimethyltin, and aminoglyco-sides.5,9,17,18 Studies have also demonstrated thata reduction of ROS attenuates noise-induced hear-ing loss.19,20 In summary, noise results in isch-emia, a condition known to affect auditory thresh-olds. This ischemia results in the generation ofROS and the damaging effects of these radicalscan be attenuated with scavengers and inhibitorsin the auditory system.

Resveratrol is found in over 70 fruits and plants,many of which are edible, such as mulberries,peanuts, and grapes. It is thought that this chem-ical is produced in response to environmentalstress or attack by pathogens including mold.Grapes contain particularly high concentrations ofresveratrol in the skin. Trans-resveratrol was firstdetected in grapevines in 1976 by Langcake andPryce,21 who found that the compound was syn-thesized by leaf tissues in response to fungal in-fection or exposure to ultraviolet light.5 Resvera-trol was brought to the public attention when itspresence in wine was reported in 1992 by Siemannand Creasy.22 The authors suggested that thiscompound might be the biologically active ingre-dient of red wine.23 The consumption of red wineis becoming increasingly popular due to the in-trigue created by the French Paradox. Despite thehigh fat diet and smoking tendencies of the pop-ulation in Southern France, there is an astonishing42% lower incidence of heart disease than thatfound in Americans.4 The effect known as theFrench Paradox has been attributed to the con-sumption of red wine. World Health Organizationdata indicate that resveratrol may be one of theactive ingredients in the wine that reduces the riskof coronary heart disease by up to 40% in red winedrinkers.1

Resveratrol has many important biologic activ-ities including: inhibition of lipid peroxidation;chelation of copper; free-radical scavenging; alter-ation of eicosanoid synthesis; inhibition of plateletaggregation; anti-inflammatory activity; vasore-laxing activity; modulation of lipid metabolism;anticancer activity; estrogenic activity; cardiopro-

tection; and neuroprotection.24-28 Bertelli et al29

investigated the absorption, the concentration indifferent organs, and the excretion of natural trans-and cis-Resveratrol after red wine oral administra-tion to rats. Their results show that prolongedadministration of red wine in the diet could lead toan increased resveratrol concentration in differenttissues even though the amount of resveratrol inthese different tissues was lower than that requiredfor pharmacological activity. This may explain itsbeneficial role against coronary heart disease.

Oxidative stress in the central nervous systemmay cause oxidation of lipoprotein particles. Theoxidized lipoproteins may damage cellular andsubcellular membranes, leading to tissue injuryand cell death. Draczynska-Lusiak et al30 haveshown that antioxidants, such as vitamins E or C,or resveratrol, protect neuronal cell damage fromoxidative stress in vitro. Results indicated thatoxidized lipoproteins may serve as an oxidativestressor, which may initiate the neuronal cell deathleading to the manifestation of Alzheimer disease.Zini et al31 studied the possible effects of resvera-trol on the mitochondrial respiratory chain in ratbrains. Resveratrol was found to decrease complexIII activity in rat brain by competition with coen-zyme Q. This property is especially interesting asthis complex is the site where reactive oxygensubstances (ROS) are generated. By decreasingthe activity of complex III, resveratrol not onlyopposes the production of ROS but also scavengesthem.31 Virgili and Contestabile14 report thatchronic administration of resveratrol to youngadult rats, significantly protects from the damagecaused by systemic injection of the excitotoxinkainic acid in the olfactory cortex and the hip-pocampus.

Several studies have demonstrated that antioxi-dants can attenuate hearing loss in various condi-tions, such as noise-induced hearing loss, ototox-icity, ischemia, and presbycusis.2,16,32-33

CONCLUSIONConsidering the importance of the biological

activities of resveratrol, along with previous stud-ies from our laboratory showing the protectiveeffects of antioxidants and dietary restriction onnoise-induced and age-induced hearing loss, thecurrent study demonstrates similar effects with


resveratrol. Thus, resveratrol appears to protectthe cochlea from acoustic trauma and ongoingstudies are being done to evaluate the efficacy ofresveratrol on age-related hearing loss.

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