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N VIVO PROTECTIVE EFFECT OF FERULIC ACID AGAINST
OISE-INDUCED HEARING LOSS IN THE GUINEA-PIG
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. R. FETONI,a C. MANCUSO,b* S. L. M. ERAMO,c

. RALLI,a R. PIACENTINI,c E. BARONE,b

. PALUDETTIa AND D. TROIANIc

Institute of Otolaryngology, Catholic University School of Medicine,argo Francesco Vito 1, 00168 Rome, Italy

Institute of Pharmacology, Catholic University School of Medicine,argo Francesco Vito 1, 00168 Rome, Italy

Institute of Human Physiology, Catholic University School of Medi-ine, Largo Francesco Vito 1, 00168 Rome, Italy

bstract—Ferulic acid (FA) is a phenolic compound whoseeuroprotective activity was extensively studied in vitro. Inhis study, we provided functional in vivo evidence that FAimits noise-induced hearing loss. Guinea-pigs exposed tocoustic trauma for 1 h exhibited a significant impairment inuditory function; this injury was evident as early as 1 dayrom noise exposure and persisted over 21 days. Ferulic acid150 mg/kg i.p. for 4 days) counteracted noise-induced hear-ng loss at days 1, 3, 7 and 21 from noise exposure. Themprovement of auditory function by FA was paralleled by aignificant reduction in oxidative stress, apoptosis and in-rease in hair cell viability in the organ of Corti. Interestinglyn the guinea-pig cochleae, the neuroprotective effect of FAas functionally related not only to its scavenging ability in

he peri-traumatic period but also to the up-regulation of theytoprotective enzyme heme oxygenase-1 (HO-1); in fact,A-induced improvement of auditory function was counter-cted by the HO inhibitor zinc-protoporphyrin-IX and paral-eled the time-course of HO-1 induction over 3–7 days. Theseesults confirm the antioxidant properties of FA as free-rad-cal scavenger and suggest a role of HO-1 as an additional

ediator against noise-induced ototoxicity. © 2010 IBRO.ublished by Elsevier Ltd. All rights reserved.

ey words: hair cells, acoustic trauma, oxidative stress, 4-hy-roxynonenal, heme-oxygenase-1.

oise-induced hearing loss (NIHL), the permanent hearingoss caused either by a single exposure to very loudounds or by repeated exposure to louder sounds over anxtended period, is a major source of hearing disability indult population worldwide (Nelson et al., 2005). Conser-

Corresponding author. Tel: �39-06-30154367; fax: �39-06-3050159.-mail address: [email protected] (C. Mancuso).bbreviations: ABR, auditory brain response; ALT, alanine transami-ase; BR, bilirubin; CO, carbon monoxide; DAPI, 4=,6-diamidino-2-henylindole; DMSO, dimethyl sulfoxide; FA, ferulic acid; h, hours;Cs, hair cells; HO-1, heme oxygenase-1; IHCs, inner hair cells; NIHL,oise-induced hearing loss; NO, nitric oxide; OHCs, outer hair cells;BS, phosphate buffered saline; PI, propidium iodide; RNS, reactiveitrogen species; ROS, reactive oxygen species; SCs, supportingells; SEM, scanning electron microscopy; TBS, tris buffered saline;hree-way ANOVA, repeated measures analysis of variance; TUNEL,

merminal deoxynucleotidyl transferase dUTP nick end labeling; ZnPP,inc-protoporphyrin-IX; 4-HNE, 4-hydroxynonenal.

306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2010.06.022

1575

ative estimates indicate that approximately 10% of the USopulation between ages 20 and 69, approximately 22illion Americans, have NIHL, accounting for about 16% ofll disabling hearing loss. In Europe, 7% of workers reporthat work affects their health in the form of hearing disor-ers and the cost of hearing loss from noise representsbout 10% of total compensation costs for occupationaliseases: the European Agency for Safety and Health atork estimates that the cost of untreated hearing loss tourope ranges from 78 to 92 billions Euros (Europeangency for Safety and Health at Work, website).

Increased production of reactive oxygen and nitrogenpecies (ROS and RNS, respectively) such as the superox-

de radical anion, nitric oxide (NO) and its redox-relatedorms, in conjunction with an imbalance of antioxidant de-enses, have been demonstrated to play a significant role inIHL as they largely participate in cellular mechanisms thatnderlie hair cell death after noise exposure and lead toensorineural hearing loss in exposed subjects (Hendersont al., 2006). After noise exposure, mitochondrial aerobicespiration increases and generates larger quantities of ROS.s ROS accumulate, the cochlea’s intrinsic antioxidant de-

enses show less effectiveness in neutralizing free radical’sction; this eventually leads to inner and largely to outer hairell death through either apoptosis or necrosis (Nicotera etl., 2004; Van De Water et al., 2004; Henderson et al., 2006).

Several molecules with antioxidant and scavengingroperties including �-tocopherol (Fetoni et al., 2004a,b;e Prell et al., 2007a), idebenone (Sergi et al., 2006; Fetonit al., 2008), the water-soluble formulation of coenzyme

10 (Fetoni et al., 2009b), glutathione (Hight et al., 2003),-acetylcisteine (Kopke et al., 2007), D-methionine (Camp-ell et al., 2007) have been tested to reduce oxidativetress-induced hair cell death after intense sound expo-ure. In this study, we focused on ferulic acid (FA, 4-hy-roxy 3-methoxycinnamic acid), a phenolic compound withntioxidant, antimicrobial, antihyperlipidemic and antihy-ertensive properties. Through its free radical scavengingroperty, largely mediated by the capability to form a res-nance-stabilized phenoxyl radical, FA was shown to beffective as a neuroprotector in several in vitro and ex vivoodels of neurodegenerative disorders such as Alzhei-er’s disease, Parkinson’s disease and cerebral ischemia/

eperfusion injury (Barone et al., 2009). In addition to airect scavenging activity, several lines of evidence dem-nstrated that FA exerts its cytoprotective effect by in-reasing cell stress response (Calabrese et al., 2008; Bar-ne et al., 2009). Among the many intracellular pathways

nvolved in the adaptive stress response activated by FA, a
ajor role is played by the inducible isoform of heme
s reserved.

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A. R. Fetoni et al. / Neuroscience 169 (2010) 1575–15881576

xygenase (HO-1), the microsomal enzyme deputed toeme catabolism (Mancuso et al., 2007; Calabrese et al.,008; Barone et al., 2009). Heme oxygenase-1 increasesell stress response by at least three mechanisms: (i)educing the amount of heme which may become pro-xidant in cells with an impaired redox metabolism, suchs under conditions of oxidative stress, (ii) producing car-on monoxide (CO), a vasoactive molecule which acti-ates many pro-survival pathways and (iii) generating biliv-rdin which is the precursor of bilirubin (BR), an endoge-ous molecule with strong antioxidant and antinitrosative

eatures (Mancuso, 2004; Mancuso et al., 2006; Mancusond Barone, 2009). HO-1 has been reported to downregu-

ate ROS in hair cells after cisplatin administration (Kim etl., 2006), and antioxidants such as ebselen attenuateisplatin induced cochlear damage by up-regulating HO-1xpression (Kim et al., 2009).

This study was designed to test the hypothesis whetherA could provide in vivo protection against noise-induced hairell loss in the guinea-pig. We evaluated the effectiveness ofA by measuring auditory brain response (ABR) thresholds,

he extent of damage with cochleogram, the magnitude ofipid peroxidation by the expression of 4-hydroxynonenal (4-NE) and the systemic inflammatory response by two wellnown biomarkers such as serum alanine transaminaseALT) and C-reactive protein. The expression of the activeorm of the caspase-3 and terminal deoxynucleotidyl trans-erase dUTP nick end labeling (TUNEL) assay as parametersf apoptotic activation and the role of HO-1 were studied.

EXPERIMENTAL PROCEDURES

nimals and drug administration

dult Hartley albino guinea-pigs (age 3 months), weighing 250–50 g, with normal Preyer’s reflex, were used in this study. All

able 1. Study design

Number of animals used for each experimental procedure. 117 animhey were divided into 9 groups. At day 1 post noise exposure they weaspase-3 immunostaining; at day 2, 14 animals were sacrificed foracrificed (19 for Western Blot, 13 for 4-HNE and HO-1 immunostainingBR tested then 29 sacrificed (16 for Western Blot, 13 for 4-HNE a
acrificed; at day 21 the remaining 24 animals were ABR tested and sacrificed
rocedures on animal use and care were conducted in accor-ance with the Laboratory of Animal Care and Use Committee ofhe Catholic University, School of Medicine of Rome, and of theuropean Communities Council Directive (86/609/EEC) and werepproved by the Italian Department of Health (Ministero dellaalute).

Ferulic acid (Sigma-Aldrich, St. Louis, MO, USA) was dilutedn dimethyl sulfoxide (DMSO) to reach the concentration of 150

g FA/450 �l DMSO; the solution was prepared freshly on a dailyasis. Zinc-protoporphyrin-IX (ZnPP, Frontier Scientific, Logan,T, USA) was diluted in 0.1 M NaOH at the stock concentration of0 mM and then diluted in saline. All chemicals were obtainedrom Sigma-Aldrich unless otherwise specified.

A total of 117 guinea-pigs were randomly divided into nineroups and sacrificed at different time points depending on thexperimental procedure(s) as shown in Table 1. Briefly, in the Controlroup, 19 animals did not receive any treatment, in the Vehicle group1 animals were injected with DMSO once daily for 4 days by i.p.oute, in the FA group, 22 animals were treated with FA (150 mg/kg.p.) for 4 days (Perluigi et al., 2006), in the Noise group 24 animalsere exposed to a pure tone sound of 120 dB at a frequency of 6Hz and in the Noise�FA group 26 animals were exposed tooise and received FA as above 1 h before and once daily for theollowing 3 days. Three animals received FA 24 h after noisexposure and then once daily for 3 days. In other experiments andditional 12 animals were divided in 3 further groups as follows:guinea-pigs were treated with ZnPP at the dose of 15 �mol/kg

.p. for 4 days (ZnPP group); in the Noise�ZnPP group 4 animalsere treated one day before and 3 days after noise exposure and

n the Noise�ZnPP�FA, another 4 animals were treated withnPP (15 �mol/kg i.p.) and FA (150 mg/kg i.p.) after the noisexposure at the same time points. In all the experiments,uinea-pigs exposed to DMSO did not show any significanthange in all the parameters measured by ABR or immuno-taining or Western blot procedures with respect to controlntreated animals and therefore the Vehicle group was consid-red as the Control group. All animals were sacrificed with a

ethal injection of Ketamine hydrochloride (75 mg/kg plus xyla-ine 20 mg/kg).

ABR tested for baseline before noise exposure (time point day 0) thenABR tested, afterwards in the same day 14 were sacrificed for activeassay; at day 3 all the remaining 89 were ABR tested and then 327 the remaining 57 animals belonging to the different nine groups wereimmunostaining) and 4 animals, treated with only ZnPP, were also

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A. R. Fetoni et al. / Neuroscience 169 (2010) 1575–1588 1577

oise exposure

s described in previous papers (Sergi et al., 2006; Fetoni et al.,008), the acoustic trauma was induced by a continuous pure tonef 6 kHz generated by a waveform generator (LAG-120B, Audioenerator, Leader Electronics Corporation, Yokohama, Japan)nd amplified by an audio amplifier (A-307R, Pioneer Electronics,ong Beach, CA, USA). As specified in Table 1, a total of 61uinea-pigs were exposed to acoustic trauma and sacrificed atifferent time points according to the different experimental pro-edures used in the present research. The animals were deeplynesthetized (Ketamine hydrochloride 25 mg/kg, xylazine 5 mg/kgnd acepromazine maleate 1.5 mg/kg body weight), placed in aound-proof room and exposed for 60 min to a 120 dB SPL pureone sound at a frequency of 6 kHz. The sound was generated by

waveform generator (LAG-120B), amplified by an audio ampli-er (A-307R) and symmetrically presented in open field by a domeweeter (TW340X0, Audax, Chateau du Loir, France) positioned0 cm in front of the animal’s head. Sound level was measuredsing a calibrated 1/4 in. microphone (Model 7017, ACO Pacificnc., Belmont, CA, USA) and a calibrated preamplifier (Acousticnterface System, ACO Pacific Inc).

uditory brainstem responses (ABR)

earing function was evaluated in all animals by measuring ABRt low (2, 4 kHz), mid (6, 12 kHz) and high (16, 20 kHz) frequen-ies at several time points before (time point day 0 in Table 1) and, 3, 7 and 21 days after noise exposure; however only a set ofnimals for each experimental group was monitored up to day 21.s reported in Table 1, 117 animals were tested prior to noisexposure and then again at day 1, 89 animals at day 3, 57 at dayand finally 24 animals at day 21 after noise exposure. The 12

nimals treated with ZnPP and the 3 animals treated with FAtarting 24 h after noise exposure underwent ABRs before noisexposure and at days 1, 3 and 7. Animals were mildly anaesthe-

ized as described above and placed in a soundproof room. Threetainless steel recording electrodes were subcutaneously insertedosterior to the tested pinna (active), vertex (reference) and con-

ralateral pinna (ground). A computer-controlled TDT System 3Tucker–Davis Technologies, Alachua, FL, USA) data acquisitionystem with real-time digital signal processing was used to recordhe ABR and generate the auditory stimulus. Tone bursts rangingrom 2 to 20 kHz (rise/fall time, 2 ms; total duration, 2 ms; repe-ition rate, 21/s) were presented monaurally in an open field using

horn tweeter (Tucker–Davis Technologies). Responses wereltered (100–3000 Hz band-pass), digitized and averaged across000 discrete samples at each frequency-level combination.hresholds were determined by increasing the intensity of the

one in 5 dB steps starting at 0 dB and increasing to 100 dB or untilhe ABR response was detected. Then, the stimulus intensity wasecreased in 5 dB steps until the latency-appropriate responseisappeared. The threshold value was defined as the lowest in-

ensity able to evoke an appropriate ABR response (Fetoni et al.,008).

mmunohistochemistry

Active caspase-3 immunostaining. To detect the expres-ion of the active form of caspase-3, a total of 14 guinea-pigs (aseported in Table 1) were deeply anesthetized and sacrificed 24 hfter noise exposure. The cochleae were quickly removed from

he skull and fixed with 10% buffered formalin for 4 h (Fetoni et al.,009b). The following procedure was performed under dim light.he cochleae were then dissected in 0.1 M phosphate bufferedaline (PBS) and permeabilized with 0.2% Triton X-100 in PBS forh at room temperature. The cochleae were rinsed twice in PBS

nd incubated overnight at 4 °C with a solution containing rabbit
rimary antibody anti-active form of caspase-3 (Chemicon Inter- 0
ational, Millipore, Billerica, MA, USA) diluted 1:200 in PBS. Spec-mens were washed twice in PBS and incubated at room temper-ture for 2 h in goat anti-rabbit secondary antibody conjugated tolexa Fluor 488 (IgG, Invitrogen, Carlsbad, CA, USA) diluted:200 in 0.1 M PBS and then washed in PBS. The specimensere double stained with DAPI (4=,6-diamidino-2-phenylindole,

nvitrogen), a DNA intercalating fluorescent probe used for visu-lization of HCs nuclei, diluted 1:500 in 0.1 M PBS for 5 min atoom temperature. After rinsing in PBS, the organs of Corti wereounted on microscope slides and coverslipped with ProLongold anti-fade reagent (Invitrogen). Cells double stained (green/lue) were identified as active caspase-3-positive cells. Countingas performed in the area of major damage and is expressed inercentage of labeled inner hair cells (IHC) and outer hair cellsOHC) cells. In all cochleae the major damage covered an areaited from 12 to 17 mm from the apex.

TUNEL assay. TUNEL (APO-BrdU™ TUNEL Assay Kit—nvitrogen) was used to detect DNA fragmentation in the nuclei ofpoptotic cells in the organ of Corti. Fourteen deeply anaesthe-ized animals, belonging to the different groups (see Table 1),ere sacrificed 48 h after noise exposure. The cochleae wereuickly removed from the skull and processed as reported in arevious paper (Fetoni et al., 2008). Assay was performed accord-

ng to manufacturer’s instructions. All the procedures were per-ormed under dim light. Briefly, the cochleae were fixed with 10%uffered formalin for 4 h. After microdissection in 0.1 M PBS,urface preparations of the organ of Corti were incubated ince-cold 70% (v/v) ethanol overnight and then incubated overnightt room temperature in freshly prepared DNA-labeling solution.he specimens were rinsed twice in the rinse buffer and thentained with Alexa Fluor 488 (IgG, Invitrogen) dye-labeled anti-rdUTP antibody for 1 h at room temperature. The organs of Cortiere double stained with PI (Propidium Iodide, 1:100) for 20 mint room temperature. After rinsing in PBS, the organs of Corti wereounted on slides as described in the previous section. Cells with

ntense yellow-labeled nuclei (red plus green) were identified aspoptotic cells. Counting was performed in the area of majoramage and is expressed in percentage of labeled IHC and OHCells.

4-Hydroxynonenal and HO-1 immunostaining. The expres-ion of both 4-HNE and HO-1 was detected respectively in theight and in the left cochleae of 26 guinea-pigs as reported inable 1. After ABR testing, the animals were deeply anesthetizednd sacrificed at day 3 (13 animals) and day 7 (13 animals). Asescribed in a previous work (Fetoni et al., 2009a), cochleae wereuickly removed from the skull and fixed with 10% buffered for-alin for 4 h. Cochleae were then dissected in 0.1 M PBS, and thergans of Corti were incubated with a blocking solution containing% fatty acid-free bovine serum albumin (BSA), 0.5% Triton X-100nd 10% rabbit serum in PBS for 1 h at room temperature. Theight specimens (for 4-HNE) were incubated overnight at 4 °C with

solution containing rabbit polyclonal anti 4-HNE primary anti-ody (rabbit Anti 4-HNE antiserum, Cat#HNE11s, Alpha Diagnos-ics, TX, USA) diluted 1:100 in PBS. The left cochlea specimensfor HO-1) were incubated overnight at 4 °C with a solution con-aining anti-HO-1 primary antibody (SPA-895, Stressgen, Annrbor, MI, USA) diluted 1:100 in PBS. Both the rabbit anti-4-HNEnd anti-HO-1 antibodies cross-reacted with guinea-pig tissue. Athe end of incubation, all specimens were washed twice in PBS,nd incubated at room temperature for 2 h in labeled conjugatedoat anti-rabbit secondary antibody (Alexa Fluor 488, IgG, Invitro-en) diluted 1:400 in 0.1 M PBS and then washed in PBS. The rightpecimens (4-HNE) were double stained with DAPI (blue fluores-ence) diluted 1:500 in PBS for 5 min at room temperature androtected from light. The left specimens (HO-1) were double stainedith PI (Invitrogen), red fluorescence, at the dilution 5 �g/ml of PI in
.1 M PBS for 15 min at room temperature and protected from

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ight. After rinsing in PBS, the organs of Corti were mounted onlides as described above. 4-hydroxynonenal positive cells of theight specimens and HO-1 positive cells of the left specimens weredentified by green fluorescence scattered over the length of thergans of Corti.

onfocal microscopy

pecimens processed for active-caspase-3 labeling, TUNEL as-ay, 4-HNE and HO-1 expression were observed with a confocalaser scanning system (TCS-SP2; Leica Microsystem, GmbH,

ezlar, Germany), equipped with an Ar/ArKr laser and a HeNeaser for 488 and 543 nm excitation. DAPI staining was imaged by-photon excitation (740 nm, �140 fs, 90 MHz) performed by anltrafast tunable mode-locked Ti: saffire laser (Chameleon, Co-erent Inc., Santa Clara, CA, USA). Images were taken at 40� or3� magnification. Z-stack series of 15–20 �m-thick were ac-uired as images of 512�512 pixels recorded at intervals of 0.5m. Positive cells were counted in segments of about 400 �m

ength each along the basilar membrane. Control experimentsere performed by omitting the primary antibodies during pro-essing of tissue randomly selected across experimental groups.o stain was present in these specimens, indicating neither spon-

aneous fluorescence nor non-specificity of the secondary anti-ody (see e.g. Fig. 4, panels D, J).

estern blot for HO-1

fter ABR measurement, as specified in Table 1, 35 guinea-pigselonging to the different groups were sacrificed by a lethal dosef anesthetic cocktail and the cochleae were quickly removed fromhe skull, the organ of Corti isolated from the surrounding bone,rozen in liquid nitrogen and stored at �80 °C until used. Samplesere then homogenized using a RIPA buffer containing 50 mMris/HCl pH 7.4 150 mM NaCl, 1% SDS, 1% Triton X-100, and 1%f a cocktail of inhibitors (1 mM PMSF, 2 mM leupeptin, 1.5 mMepstatin A, 80 mM aprotinin, 4 mM pepstatin, 1.4 mM E-64, 104M AEBSF, 1 mM EDTA, 0.2 mM Na3VO4, and 0.1 mM NaF).fter centrifugation (15 min to 15,000 rpm to 4 °C), supernatantsere collected and protein concentration measured using theicro BCA kit (Pierce, Rockford, IL, USA). For each sample, 40g of proteins were separated by SDS-polyacrylamide gel elec-

rophoresis (SDS-PAGE) on 10% gel. Proteins were then trans-erred onto nitrocellulose membranes using a solution containing0 mM Tris/HCl, 380 mM glycine and 20% methanol and stainedith Ponceau S to ensure protein transfer (ICN Biochemicals, OH,SA). Nonspecific binding sites were blocked with 5% dry milk inris buffered saline (TTBS, 20 mM Tris, 500 mM NaCl, and 0.05%ween-20) and the membranes were incubated overnight at 4°Cith the primary antibody (anti HO-1, SPA-895, 1:2000, Stress-en) diluted in 3% BSA/0.05% NaN3/TTBS. After 5 rinses inuffer, membranes were incubated for 1 h at room temperatureith a horseradish peroxidase conjugated anti-rabbit IgG second-ry antibody (Promega, Madison, WI, USA), diluted 1:5000 in.5% dry milk/TTBS, and then developed using enhanced chemi-

uminescence reagents (ECL, Amersham Biosciences, Bucking-amshire, UK). The immunoreacted bands were visualized byxposing membranes to Kodak X-OMAT films. Equal protein load-

ng among individual lanes was confirmed after stripping the mem-ranes with Restore Western blot Stripping buffer (Thermo Fishercientific, Waltham, MA, USA) by re-probing the membranes withn anti-�-tubulin rabbit monoclonal antibody at 1:1000 dilutionThermo Fisher Scientific, Rockford, IL, USA). This anti-rabbitntibody cross-reacted with guinea-pig tissues. The obtained filmsere evaluated for densitometry by means of an optical computedystem (Epson Perfection, 1240U Photo). Values are expresseds HO-1/�-tubulin ratio and normalized with the value in vehicle-retreated group. The experiments were repeated three times and
n inter-experiment variability of less than 10% was found. n
canning electron microscopy (SEM)

fter the day 21 final ABR recordings, 15 animals belonging to theifferent groups were deeply anaesthetized and sacrificed; theochleae were removed and processed for SEM as previouslyescribed (Fetoni et al., 2004a). Each specimen was viewed andhotographed with a Zeiss Supra 50 Field Emission SEM appa-atus (Carl Zeiss Inc., Göttingen, Germany). Quantitative SEMbservations of the surface morphology of the organ of Corti wereerformed by counting the number of missing hair cells (HCs) fromhe apex to the base of the cochlea. For cell counts, the wholeochlea was divided into 20 segments (1 mm lengths of basilarembrane) which contain an expected number of 100 IHCs and25�3 OHCs in each segment (100%) (Wang et al., 2002). Theesults of these HCs counts were expressed as the percentage ofemaining HCs in each row of IHCs and OHCs over the entireength of the cochlea. A hair cell was counted as missing whenhere was a complete disappearance of the cell and if the stereo-iliary bundle was absent or the stereocilia of the bundle wereompletely fused or totally disarrayed. All the counts were per-ormed by a blinded investigator.

nzyme activities in serum

spartic transaminase (AST) was measured in guinea-pig serumy a modification of the method described by Saris (1978),hereas ALT determination was based on a modification of therocedure by Bergmeyer et al. (1978) and on the method de-cribed by Wroblewski and Ladue (1956). C-reactive protein waseasured in guinea-pig serum by using an ELISA kit (USCN Lifecience Inc., Wuhan, China) as per manufacturer’s protocol.

tatistical analysis

ll the results are presented as means�standard error of n rep-icates per group. Differences were assessed using the analysis ofariance (ABR data: group�frequency�day, three-way ANOVAith repeated measures; Cochleogram data: group�HCs type�

ocation along 20 mm segments, three-way ANOVA with repeatedeasures). Post hoc comparisons were assessed with Tukey’s

est (Statistica, Statsoft, Tulsa, OK, USA). P-value of �0.05 wasonsidered significant.

RESULTS

uditory function evaluation

BRs were recorded in each animal before noise exposurend after 1, 3, 7 and 21 days (Fig. 1). A total of 117 animalsere used and ABR testing was performed in all animals,owever not all animals could be ABR tested at the fiveime points because some animals from each group weresed for the immunostaining and Western blot proceduressee Table 1). All ABR data are expressed in terms ofhreshold shift, which represents the difference betweenhe pre-noise and post-noise exposure value in each ani-al for each group. Consistent with previous data, thereatest hearing loss occurred in the 6–12 kHz regionentered around the frequency of acoustic trauma (Fetonit al., 2004a,b, 2009a,b; Sergi et al., 2006).

Baseline ABR thresholds (day 0) did not differ amonghe 9 assigned treatment groups consistent with previousata from our laboratory. The administration of the vehicleMSO did not alter the ABR values with respect to un-

reated guinea-pigs either in the presence or absence of
oise exposure and at each time point and frequency (data

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ot shown). In the noise exposed animals (Fig. 1A), 1 dayfter noise exposure the threshold shift was elevated tobout 40–50 dB for mid and high frequencies and to0–40 dB for the low frequencies. Three days after thecoustic trauma, there was a small partial recovery in theoise group and the average threshold shift decreasedpproximately 10 dB for high frequencies, without differ-nces for mid and low frequencies. At day 7, an additional0 dB recovery of thresholds was detected, and all fre-uencies were involved with a greater attenuation in theid and high frequency range. Over the following 2 weeks

day 21 measurement), thresholds continued to recover toeach a final threshold shift of around 15–25 dB for low,id, and high frequencies.

The FA administrations were started according to tworocedures: 24 h after the acoustic trauma (Noise�FA4 h post) and then once daily for the next 3 days or 1 hefore the acoustic trauma and then once daily for the nextdays (Noise�FA group). In Noise�FA 24 h post, ABR

nalysis, carried out up to day 7, revealed no differences inhreshold shifts with respect to the Noise group (data nothown). On the contrary, FA administered before thecoustic trauma (Noise�FA group) attenuated the eleva-ion of hearing thresholds. Fig. 1B shows that this improve-ent was clearly seen as early as day 1; the threshold shift

anged from 10 to 30 dB, mid/high frequencies were mostlyffected. At day 3 after acoustic trauma, low frequencieshresholds recovered almost completely and the thresholdhift for mid and high frequencies decreased to about5–20 dB. At day 7, thresholds continued to recover bybout 10–15 dB, threshold shifts for low (2–4 kHz) andigh (16–20 kHz) frequencies decreased to about 5 dBnd for mid frequencies (6–12 kHz) reached a value ofbout 10–15 dB. These values of threshold shift repre-ented the final hearing loss in the Noise�FA group ashere was no further improvement (except for the 12 kHzrequency) over the next 2 weeks (21 day measurement).

ig. 1. The graphs show the threshold shift values in dB (mean�stanefore acoustic trauma and at days 1 (squares), 3 (circles), 7 (trianglesoise group the maximal threshold shift ranged between 40 and 45 dBay 21, reached progressively over the 3 weeks of testing, was of 20aximal threshold shift was 30 dB; in this group complete recovery of tdB) whereas for frequencies of 6–12 kHz a threshold shift of about 1

nalysis revealed significant differences between or within groups, dasterisk are the significant differences among days within each group

ummarizing, in the Noise�FA group protection was o

learly detected at day 1 after noise exposure, the protec-ive effect progressed at days 3 and 7 when it was almostompleted.

Statistical analysis was performed on the ABR databtained in the Noise and Noise�FA groups and signifi-ant differences were found between or within groups,ays and frequencies (three-way ANOVA). The thresholdhift values in the FA protected group were significantlyttenuated with respect to the values of the Noise group:ignificant interactions were found between groups (F(1–12)�19.8 P�0.001), between groups and time points (F(3–36)�.04; P�0.05) and between time points and measuredrequencies (F(18–216)�4.65; P�0.001). The thresholdhift values were significantly attenuated throughout theourse of days: post hoc analysis showed that in the Noiseroup the difference was significant between each timeoint (P�0.001) whereas in the Noise�FA group the sig-ificance was found between day 1 and day 3, betweenay 3 and day 7 (P�0.001). The extent of threshold shiftlevation in both groups significantly increased from low toigh frequencies (post hoc P�0.001) but the increase inhe Noise�FA group at all frequencies was significantlyttenuated as compared to the Noise group (post hocnalysis P�0.001).

A mediated inhibition of apoptosis in hair cellsHCs)

s shown in Fig. 2, active caspase-3 immunostaining inCs was primarily detected in the nuclei. Caspase-3 is aytosolic protein, whose activation could be related to itsuclear translocation (Kamada et al., 2005). Accordingly,

n our experimental system the anti-active-caspase-3 an-ibody detected a remarkable nuclear staining. Confocalnalysis (Z-stack) demonstrates that active caspase-3 la-eling was primarily located in the nuclei (Fig. 2A1–F1).fter 24 h, the greatest number of activated nuclei was

r) corresponding to the differences between the recordings measured(diamonds) of Noise group (A) and Noise�FA group (B). Note: in theencies of 6–20 kHz at day 1 and the spontaneous partial recovery atreas in the Noise�FA group, at the same frequencies and day, the

s for low and high frequencies occurred at day 7 (threshold shift aboutsisted and no further improvement was obtained at day 21. Statisticalquencies (three-way ANOVA with repeated measures). Indicated byled by post hoc analysis (* P�0.001).

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oise and Noise�FA groups. In the vehicle group, as inhe control and FA exposed animals, activated caspase-3as not observed in the HCs (data not shown, see ref.etoni et al., 2009b). In the cochleae of the Noise group,

he number of caspase-3-positive nuclei was significantlyreater than in Noise�FA treated animals. In fact, in the

ig. 2. Confocal images of surface preparations of the damaged areaspase-3. Nuclei were identified in blue fluorescence by DAPI (A, D)B, E). In panels C, F merged images are shown. In the Noise group (Ahird row with a decreasing gradient from the third to the first row. Supevealed the nuclear caspase-3 labeling in the IHCs, OHCs and outerhe second row and IHCs (arrow heads in panel C1). In the Noise�Fecreased in the same region. The higher magnification images (D1–Fead in panel F1) and supporting cells. Confocal XYZ images of theemonstrate that active caspase-3 was primarily located in the nuclei o

n this figure legend, the reader is referred to the Web version of this

everely damaged area, corresponding to 12–17 mm from d

he apex of the cochlea, strong active caspase-3 fluores-ence was observed in almost all OHCs in the third rowith a decreasing number of cells from the third to the first

ow (Fig. 2B). In the Noise group a number of IHCs showedositive active caspase-3 nuclei (Fig. 2A–C). The occur-ence of active caspase-3-positive nuclei was significantly

oise and Noise�FA group cochleae stained for detection of activective caspase-3 immunoreactivity was labeled in green fluorescence

ve caspase-3-positive nuclei were observed in almost all OHCs in thend IHCs were also stained. The higher magnification images (A1–C1)pporting cells. Note a few active caspase-3-stained OHCs located inae (D–F) the number of caspase-3-positive nuclei was significantly

hat active caspase-3 was expressed in the nuclei of few OHCs (arrow(A1–C1) and (D1–F1) (Z-sections refer to the dashed lines) further

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oise�FA treated animals and only a few OHCs, occa-ional IHCs and some supporting cells showed signs ofctive caspase-3 fluorescence, a greater number of sur-iving OHCs and IHCs appeared in blue fluorescence (Fig.D–F). The percentage of active caspase-3-positive OHCsnd IHCs in the area corresponding to the major damageas 63.6�4.7% in noise-exposed cochleae: this value wastrongly reduced in Noise�FA treated cochleae (2.8�.6%).

Cochleae from noise-exposed animals showed TUNEL-ositive labeled nuclei within OHCs and non-sensory cells

n the region of the cochlea corresponding to the severeamage (Fig. 3A). Treatment with FA decreased the num-er of apoptotic cells (Fig. 3B). A decreasing number of

ig. 3. Confocal images of surface preparations of a representativeamaged area from Noise group (panel A) and Noise�FA grouppanel B) cochleae stained for the detection of apoptosis using theUNEL assay. Counting considered cells presenting the double stain-

ng and the apoptotic TUNEL-positive nuclei appeared in yellow. Theochleae from Noise group (panel A) showed apoptotic nuclei withinhe OHCs and supporting cells (SCs) in the region of the cochleaorresponding to the severe damaged area of the basal turn. In theegion of the inner pillar cells, where nonspecific green fluorescenceppears, there was no double staining with PI indicating no apoptoticrocess in the pillar cells. A significant decrease of yellow fluorescentuclei, representative of apoptotic cells, was observed in the sameegion of all cochleae of Noise�FA group (panel B). Panels A1, B1

how the enlargements of the regions of interest delineated with boxesn (panels A, B) show the nuclear fragmentation in the third and secondows. No IHCs were double stained in this sample. For interpretation of

(he references to color in this figure legend, the reader is referred tohe Web version of this article.

UNEL-positive nuclei were detected in the second andhird turns of the cochlea in all specimens. In cochleae ofnimals treated with FA positive OHC nuclei were rarelybserved in the basal turn corresponding to 12–17 mmrom the apex of the cochlea. TUNEL-positive OHC andHC nuclei in the severely damaged area were countednd the percentage of TUNEL-positive labeled nuclei wasalculated for each group. In noise exposed cochleae8.7�7.7% of HC nuclei were TUNEL-positive versus.8�3.4% for FA treated cochleae (Noise�FA group). NoUNEL-positive nuclei were observed in control, vehiclend FA animals (data not shown, see ref. Fetoni et al.,009b).

A mediated protection from oxidative stress

t is well known that lipid oxidation is a common hallmark ofxidative stress. Among the biomarkers of lipid peroxida-ion, 4-HNE is one of the more sensitive and widely used ineveral in vitro and in vivo experimental systems. A fainttaining was observed in animals treated with vehicle (Fig.A). There were no observable differences in immunocy-ochemical appearance among vehicle, control and FAochleae (data not shown).

As shown in Fig. 4B, C, the organ of Corti from noise-xposed guinea-pigs underwent oxidative stress as dem-nstrated by the marked increase in 4-HNE immunostain-

ng at days 3 and 7. Importantly, this pro-oxidant effect dueo noise exposure was counteracted by the administrationf FA, as shown in Fig. 4E, F, thus corroborating thentioxidant effect of this phenolic acid. A strong immuno-eactivity for 4-HNE was detected in almost all OHCs in theeverely damaged area, located 12–17 mm from the apexf the cochlea. Few 4-HNE-positive IHCs were detectedver the length of the organ of Corti. At day 7 after noisexposure 4-HNE remained strongly expressed with a de-reasing gradient from the third to the first row of OHCsFig. 4C). On the contrary, organs of Corti from noise-xposed animals treated with FA, exhibited at both day 3nd 7 only few OHCs positive for 4-HNE, mainly localized

n the third row of OHCs (Fig. 4E, F respectively).To determine whether noise exposure caused any sys-

emic inflammatory response, hepatic ALT and serum C-eactive protein were measured and no differences wereound with respect to controls (mean values: Control 41I/L; Noise 3 days, 38 UI/L; Noise 7 days 39 UI/L) or serum-reactive protein (mean values: Control 100%; Noise 3ays 95%; Noise 7 days 93%).

air cells viability

EM images and cochleograms are shown in Fig. 5. Inoise-exposed guinea-pigs, massive OHCs loss and slightHCs disappearance were restricted to a small area lo-ated 12–17 mm from the apex of the cochlea with aecreasing pattern in the transitional area. In detail, mostf hair cell losses were found in the first row of OHCs ando a lesser extent in the second and third OHC rows (Fig.A). The number of OHCs loss was reduced in the co-hleae of the Noise�FA group with respect to Noise group
Fig. 5B). Absent cells and cells with stereocilia damage

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ig. 4. Representative confocal images of immunostaining for 4-HNE and HO-1 of surface preparation of the organ of Corti showing the parallelismetween the decrease of oxidative stress and the increase of HO-1 antioxidant effect. Staining was absent when the primary antibodies were omitted

ndicating neither spontaneous fluorescence nor non-specificity of the secondary antibody (D, J). In the upper panels, anti 4-HNE staining wasdentified by green fluorescence and nuclei were counterstained in blue with DAPI. In the lower panels, anti HO-1 were stained in green and nucleiere counterstained in red with PI. In basal turns of vehicle group cochleae, corresponding to the most damaged area, a faint staining for 4-HNE wasbserved (Panel A). In Noise group cochlea (panel B), increased 4-HNE immunofluorescence was observed in almost all OHCs. Few IHCs showedcytosolic expression of 4-HNE. Inset B1 shows the enlargement of the region delineated by the box and identifies specific cellular staining for 4-HNE.anel C: the expression of 4-HNE remained clearly detectable at day 7 after noise exposure. Panel E: the number of OHCs showing 4-HNE

mmunofluoresce was significantly decreased in the same damaged region of FA-treated animals and immunoreactivity was observed mainly in thehird row of OHCs. Panel F: at day 7 after noise exposure in cochleae from the Noise�FA a further reduction of 4-HNE expression was observed. Inhe lower panels, anti HO-1 was stained in green and nuclei were counterstained in red by PI. In Panel G a weak staining for HO-1 was observed inehicle cochleae. Panel H: the cochleae from Noise group (day 3) exhibited a remarkable HO-1 immunoreactivity in the region corresponding to theevere damaged area (12–17 mm from the apex). Panel I: at day 7 after noise exposure, a less intense immunostaining was observed in the OHCsf the same area. In cochleae from Noise�FA group strong HO-1 immunoreactivity was present at days 3 (panel K) and 7 (panel L), although less

ntense than day 3. Insets H1, K1 show the enlargements of the regions of interest delineated with boxes in (panels H, K). Nonspecific staining wassually observed in the region of the outer sulcus (see samples C and I). D3, day 3 from noise exposure; D7, day 7 from noise exposure. For

nterpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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ere counted and the mean hair loss is quantified in theower panels of Fig. 5. Three-way ANOVA (group�HCsype�location along the cochlea) demonstrated significantifferences between groups (F(1–6)�33.09; P�0.001), be-ween IHCs and OHCs (F(1–6)�415.33; P�0.001) andmong each segment along the entire length of the basilarembrane (F(19–114)�84.90; P�0.001). Post hoc analysis

evealed a significant differences between IHCs and allHCs within each group (P�0.001). Post hoc comparison

or OHCs factor between Noise and Noise�FA groupshowed that significant differences were present in therea located 12 and 17 mm from the apex of the cochlea,owever no significance was found for IHCs.

A cytoprotection through HO-1

s shown in Fig. 6, the protective effect of FA on guinea-ig auditory function was inhibited by the concomitantdministration of ZnPP (15 �mol/kg i.p. once daily for 4ays), a well known inhibitor of HO activity. Three-wayNOVA demonstrated a significant difference among the

our groups: Noise, Noise�ZnPP, Noise�ZnPP�FA andoise�FA across time points (F(6,36)�2.62; P�0.05),hereas ZnPP alone did not exhibit any significant effectn guinea-pig auditory function (data not shown). At day 1Fig. 6A) the Noise�ZnPP animals showed a non signifi-ant increase of threshold shift as compared to Noiseroup. The administration of FA in the Noise�ZnPP group

ig. 5. Typical SEM images of Noise (panel A) and Noise�FA (panelotted as percentage with standard error of survival IHCs and OHCs v

f the stereociliary bundle was absent (arrowhead in panel A) or the sten panel A). In (B), a few HCs were absent and some OHCs and IHCsnner pillar cells (IPs). The cochleograms (panels C) shows that no difetween Noise and Noise�FA groups. (Panel D) shows that the OHCost hoc analysis for OHCs between Noise and Noise�FA was signi

Noise�ZnPP�FA) decreased the threshold shift slightly (

ut significantly (P�0.05) as compared to the Noise group;n the contrary in the protected animals without ZnPPNoise�FA group), at this time point, the threshold shifthowed early signs of improvement and the differenceetween this group and each of the other three was sig-ificant (P�0.001). At day 3 (Fig. 6B) the effects of FA inhe Noise�ZnPP�FA group were inhibited and thresholdhift values showed non significant variations betweenoise�ZnPP and Noise�ZnPP�FA however a significantap remained between each of these groups and theoise Group. At day 7 (Fig. 6C), the differences between

he groups treated with ZnPP and the Noise group wereot significant. On the contrary, in the Noise�FA group the

nitial threshold shifts were further attenuated as describedbove. Post hoc analysis did not reveal any significancemong groups or frequencies at day 7, the only signifi-ance was versus the Noise�FA group (P�0.001).

The evidence that ZnPP counteracted the protectiveffect of FA on noise-induced hearing loss, suggested aole for HO in the cytoprotection mediated by the phenoliccid and prompted us to investigate the pattern of expres-ion of HO-1 in this experimental model. In order to inves-igate the contribution of HO-1 in the cytoprotective effectf FA, cochleae from guinea-pigs exposed to noise in theresence or absence of FA were processed for immuno-istochemistry with anti-HO-1 antibody. Immunostainingor cochlear HO-1 confirmed the Western Blot findings

ected cochleae and related cochleograms (panels C, D respectively)tance from the apex of the cochlea. A hair cell was counted as missingf the bundle were completely fused or totally disarrayed (white arrowsaged. The IHC stereocilia (black arrow in B) are shown on top of the

were found for IHCs along the entire length of the basilar membraneffected region corresponded to the middle-basal turns of the cochlea.�0.05) at the location 12–17 mm from the apex of the cochlea.

l B) protersus disreocilia owere damferences

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tronger in Noise�FA animals as compared to control and

ig. 6. The graphs show the threshold shift values (mean�standardrror) measured in Noise, Noise�ZnPP, Noise�ZnPP�FA andoise�FA groups. Panel A: at day 1 the Noise�ZnPP and theoise�ZnPP�FA animals showed respectively an increase and de-rease of threshold shift as compared to the Noise group; in theoise�FA group the attenuation of threshold shift was evident. Panel: at day 3 not significant variations were observed betweenoise�ZnPP and Noise�ZnPP�FA however a significant difference

emained among these two groups and the Noise Group. Panel C: atay 7, the differences among all the groups treated with ZnPP and theoise group were not significant, while threshold shift measures were

urther decreased in animals of Noise�FA group.

oise group. In the Vehicle group (Fig. 4G) as in the o

ontrols (data not shown) a weak HO-1 immunoreactivityas observed in the OHCs over the length of the organ oforti. In the FA group also a slight immunolabeling wasetected in the OHCs (data not shown). Immunofluores-ence for HO-1 was increased in both Noise andoise�FA groups at day 3 from noise exposure as com-ared to controls. However, in the Noise�FA group the la-eling was markedly intense. In both Noise and Noise�FAroups the HO-1 immunofluorescence in OHCs decreasedt day 7, the staining in the Noise�FA animals remainingore elevated as compared to Noise group. Interestingly,O-1 immunoreactivity in the animals exposed to noisend treated with FA showed the same spatial distributions compared to the 4-HNE data in the Noise exposednimals, namely a strong staining in OHCs of the basalart of the cochlea corresponded to the severely damagedrea with a decrease in the middle and apical turns. In theoise�FA animals at day 3 the reduced expression of-HNE was paralleled by a higher immunoreactivity forO-1 (Fig. 4E vs. K). Moreover, in this group at day 7 aimilar trend of expression for both 4-HNE and HO-1 wasetected (Fig. 4F vs. L).

The involvement of HO-1 in the cytoprotective effect ofA was also demonstrated by Western Blot. HO-1 immu-oblotting (Fig. 7) were performed on days 3 and 7 forach experimental group (see Table 1) and �-tubulin wassed as reference. Western Blot analysis revealed thatO-1 is expressed in the cochleae of control animals andignal was maintained stable in each time point (data nothown) and therefore in the representative gel shown inig. 7A a single lane is shown.

In Noise group a strong HO-1 signal amounting to00% and 50% versus controls was found 3 and 7 daysfter noise exposure, respectively. Ferulic acid potentiatedoise-induced HO-1 over-expression of about 50% and0% after 3 and 7 days, respectively. An intriguing piece ofvidence is that a different pattern of HO-1 induction wasound depending on the time of FA administration. Ashown in Fig. 7B, guinea-pigs exposed to FA for 3 daystarting 1 day after noise exposure did not exhibit any

ncrease in HO-1 expression up to 7 days from noisexposure. On the contrary, when FA was given 1 h beforeoise and for the next 3 days, HO-1 expression at day 7as markedly increased.

The antioxidant and cytoprotective effect of the FA/O-1 pathway in this experimental system was furtherorroborated by inhibiting HO activity. ZnPP not only ex-cerbated noise-induced hearing loss (Fig. 6) but also

ncreased systemic pro-inflammatory markers the lattereing partly reverted by the co-administration of FA (meanalues of ALT 3 days after noise exposure: Control 40 UI/L;oise 39 UI/L; Noise�ZnPP 61 UI/L; Noise�ZnPP�FA 48I/L. Mean values of C-reactive protein 3 days after noisexposure: Control 100%; Noise 97%; Noise�ZnPP 278%;oise�ZnPP�FA 189%). Finally, the functional and mo-

ecular outcomes secondary to ZnPP treatment were re-ated only to its inhibition of HO activity without any effect
n HO-1 protein expression (Fig. 7C).

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DISCUSSION

n this paper, we investigated the cytoprotective effects ofA on the auditory function in the guinea-pig exposed tooise trauma and demonstrated that such an effect can beediated by both the direct free radical scavenging activityf FA and the activation of HO-1. Namely, in this model ofcoustic trauma (6 kHz, 120 dB), it was observed that FA

reatment: (a) decreased ABR threshold shift at all timeoints after noise exposure, (b) decreased oxidative stressiomarkers as shown by decreased expression of 4-HNE

aken as a marker of lipid peroxidation, (c) decreased signsf apoptosis as evidenced by both active caspase-3 andUNEL assays, (d) increased survival of hair cells asvidenced by SEM; hair cell loss count well correlated with

hreshold shift data as evidenced by cochleograms andBR values, (e) increased expression of HO-1 at 3 and 7ays from noise exposure.

Traditionally, prevention of NIHL has been addressedy providing wearable hearing protection and reducingoise emissions. However, in the last decades the in-reased knowledge of the cellular and biochemical basis ofIHL have lead to a new therapeutic approach using an-

ioxidants, N-methyl-D-aspartate (NMDA) antagonists,aspase or cell death inhibitors, and growth factors (Hen-erson et al., 2006; Le Prell et al., 2007b). Among thesetrategies, the use of selected antioxidants to neutralizeOS and inhibit cell death seems to be the most rational

ig. 7. A representative Western Blot documenting the FA-inducedcoustic trauma. A marked increase in HO-1 expression was founoise-induced HO-1 over-expression at both time points (panel A).uinea-pigs was higher when the phenolic acid was given 1 h before an4 h after noise exposure (N�FA post) (panel B). The inhibitor of HOxposed to noise and treated with FA (panel C).

pproach. Many antioxidant agents have been tested suc- t

essfully, such as the glutathione, N-acetylcysteine and-methionine, GluR-phenylisopropyladenosine, vitamin C,itamin A and vitamin E (Henderson et al., 2006; Campbellt al., 2007; Kopke et al., 2007; Le Prell et al., 2007a). Theechanism of protection exerted by FA in NIHL is consis-

ent with the protection observed by using the other anti-xidant molecules.

Several reports have shown that FA, a dietary phenolbundant in vegetables (artichokes, eggplants) and maizerain, counteracts oxidative damage through its directOS/RNS scavenging activity as well as via the inductionf pro-survival systems such as HO-1. According to theeculiar pharmacokinetics of (poly) phenols, FA has a veryoor bioavailability if given per os and its amount in tissues

s considered negligible (Barone et al., 2009). For thiseason, in many preclinical studies in rodents, FA wasdministered at “pharmacological doses,” namely at largeroses than those used per os but through a parentheraloute of administration and for a limited time (Lesca, 1983;aurya et al., 2005; Joshi et al., 2006; Perluigi et al., 2006;heng et al., 2008a). The same approach was used in theurrent study and FA was administered at the dose of 150g/kg i.p. once daily for 4 days.

The reason why FA rather than its ethyl ester washosen for this study depends on both the route of admin-

stration and the lipophilicity of the molecule. In fact, wheniven i.p. FA skips the first-pass metabolism due to intes-

n of heme oxygenase-1 (HO-1) in guinea-pig cochleae exposed tooth 3 and 7 days from noise exposure and FA further increased

-induced overexpression of HO-1 in the cochlea of noise exposedafter noise exposure (N�FA pre-post) than the administration starting

nPP did not affect HO-1 overexpression in the cochlea of guinea-pigs

modulatiod after bThe FAd 3 days

inal and hepatic degradation, reaches the blood-stream

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ery quickly and arrives to tissues. Therefore, the lipophi-icity of the molecule is not a limiting issue. Furthermore,lmost all the ex vivo studies were performed by usingerulic acid ethyl ester but only few of them addressed theotential therapeutic effect of the parent compound.

Considering that different mechanisms are involved inhe oxidative pathway and several molecules show effec-iveness in providing improvement of hearing threshold, itan be suggested that also each antioxidant molecule mayct with different targets. An intriguing part of this study ishe convergence of the functional data obtained with thedministration of FA and the regulation of HO-1 expres-ion. In fact, during the last 5 years, several in vitro and exivo studies documented a strong contribution of HO-1 inhe cytoprotective effects of FA. From a mechanistic pointf view, HO-1 is an early gene, namely its over-expression

s one of the earlier cellular events in case of oxidative/itrosative stress induction. However, if the cell is treatedith antioxidants to counteract pro-oxidant conditions,O-1 expression could further increase thus strengthening

he adaptive stress response (Calabrese et al., 2008; Bar-ne et al., 2009). The mechanism(s) through which HO-1

nduction could favor the cytoprotective effect of FA areelated to the ability of this enzyme to (i) degrade heme,hich in the presence of ROS generates the perferryl

adical which, in turn, stimulates lipid peroxidation (Ostdalt al., 1997; Klouche et al., 2004) and (ii) produce biliver-in, the precursor of the strong antioxidant and antinitro-ative molecule bilirubin (BR) (Mancuso, 2004; Mancusot al., 2006; Mancuso and Barone, 2009). This paradigmas also confirmed in our study. In fact, ZnPP given by i.p.

oute, through the inhibition of the antioxidant HO activity,aused a systemic inflammatory response in noise-ex-osed guinea-pigs as demonstrated by a significant in-rease in both hepatic transaminase ALT and C-reactiverotein serum levels, which was at least in part reverted byhe administration of FA. Previous researches demon-trated that noise exposure increased both ROS and NOormation in guinea-pig cochlea; time course analysishowed that free radical formation is very high as early as2–24 h from noise exposure and is maintained up to 7ays (Ohlemiller et al., 1999; Chen et al., 2005; Cheng etl., 2008b). Both ROS and NO formation increases lipideroxidation and protein nitration demonstrating functionalonsequences for cells (Ohinata et al., 2000, 2003; Ya-ashita et al., 2005). The results shown in this study are inood agreement with the above mentioned pattern of freeadical formation. Consistent with a recent paper (Fetoni etl., 2009a), free radical-induced lipid peroxidation, evalu-ted by measuring 4-HNE labeling, was demonstratedfter 3 and 7 days from noise exposure and was markedlyeduced by the co-administration of FA. Similarly, the ac-ivation of the apoptotic cascade, evaluated as activeaspase-3 and TUNEL assays, peaked 24–48 h fromoise exposure, even if previous data demonstrated thatrogrammed cell death is a precocious event starting asarly as 12 h from acoustic trauma (Hu et al., 2002; Fetonit al., 2009b). Moreover under pro-oxidant conditions the
dministration of FA exerted an important otoprotective i
ctivity and rescued cells from apoptotic death. It is note-orthy to underline that the pro-oxidant effect of noisexposure was restricted to the cochlea and did not haveny systemic repercussion as demonstrated by the hepaticLT and C-reactive protein serum levels. Keeping this inind, it is plausible to hypothesize that HO-1 over-expres-

ion, seen at 3 and 7 days from noise exposure in both theoise and Noise�FA groups, is an adaptive response of

he cells to oxidative stress and apoptosis. Once again,hese molecular events parallel the functional evidence. Inact, FA-related amelioration of auditory function reacheshe maximum 3 and 7 days after noise exposure matchingith the HO-1 over-expression pattern. This hypothesis isorroborated by the functional data showing that ZnPP, an

nhibitor of HO activity, when given concomitantly with FA,ignificantly counteracted FA-induced improvement of au-itory function either 3 or 7 days from noise exposure, that

s at the same time points of HO-1 induction. The onlyiscrepancy between functional and molecular data can beound when the antioxidant activity of FA was studied atarlier time-points. In fact, our functional data demon-trated that FA ameliorates noise-induced damage asarly as 24 h, a time point in which HO-1 over-expressionas not demonstrated in our experimental system (dataot shown). This last finding, together with the lack of HO-1

nduction at this time point, supports the possibility of aeuroprotective activity of FA related to its direct free-adical scavenging activity (Barone et al., 2009). On theontrary, after 3–7 days, when noise-induced pro-oxidantonditions still persist, HO-1 is over-expressed and thisffect is potentiated by FA. It is noteworthy to mention thatur study provides new evidence in support of the func-ional role for HO-1 together with HO-2 in the auditoryeurotransmission and otoprotection in the guinea-pigWatanabe et al., 2003; Matsunobu et al., 2009).

Another interesting result, consistent with previousorks, concerns the timing of protection. Recently, ourroup has studied the effectiveness of a coenzyme Qynthetic analogue idebenone (Sergi et al., 2006; Fetoni etl., 2008) and its water soluble formulation coenzyme Q-er (Fetoni et al., 2009b) in the same experimental modelemonstrating that the “therapeutic window” for successfulntioxidant approach in NIHL occurs within the first 3 daysfter noise exposure as reported also by other authorsYamashita et al., 2005). This hypothesis is strengthenedy the results shown in this paper. In fact, the lack offficacy of FA administered 24 h after noise exposure, is inood agreement with the general idea that natural antioxi-ants achieve their best cytoprotective capacity if givenefore and soon after the stressor (Ohinata et al., 2000;amashita et al., 2005). The results with systemic drugdministration at higher doses of antioxidants for a shortime in the peri-trauma period are encouraging for clinicalpplication.

Finally, the results shown in this study are in goodgreement with our previous findings in the same NIHLodel. In fact, vitamin E as well as coenzyme Q10 and its

ynthetic analogs (Q-ter and idebenone) attenuated noise-
nduced damage and exerted a cytoprotective effect on

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HCs owing to their well-known free radical scavengerctivity (Fetoni et al., 2008, 2009b). However, by compar-

ng the different mechanism(s) of action of these dietaryntioxidants against acoustic trauma, it can be underlined

hat vitamin E is a fat-soluble hydrogen atom donor and anfficient chain-breaker, coenzyme Q10 plays a role in mi-ochondrial bioenergetics as electron carrier whereas FAehaves not only as a free radical scavenger but also asn inducer of antioxidant genes (Calabrese et al., 2008;arone et al., 2009). In this light the dual action of FA coulde considered as a composite cytoprotective mechanismgainst free radical induced damage.

To the best of our knowledge, this preclinical studyeports for the first, a therapeutic activity of systemic FA inreventing noise-induced auditory loss and provides mo-

ecular evidence for a possible involvement of HO-1 inuch neuroprotective effect.

cknowledgments—This work was supported by “Fondi di Ate-eo” from the Catholic University to A.R.F, C.M. and D.T. Theonfocal analysis has been performed at the Labcemi, UCSC,ome. The Authors are grateful to Prof. Alvaro Mordente forerum enzyme activity assays and to Dr. Raffaella Siciliano for herontribution to prepare figures.

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(Accepted 10 June 2010)(Available online 20 June 2010)

in vivo protective effect of ferulic acid against noise ... · in dimethyl sulfoxide (dmso) to...

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