Medical
ozone
therapy
for
the
inner
ear
acoustic
trauma
Alper
Yenigun
a,*,
Fadlullah
Aksoy
b,
Remzi
Dogan
b,
Fahrettin
Yilmaz
c,
Bayram
Veyseller
b,
Orhan
Ozturan
b,
Burak
Ozturk
da
KaramanStateHospital,OtorhinolaryngologyClinic,Karaman,Turkey
b
BezmialemVakifUniversity,DepartmentofOtorhinolaryngology,Istanbul,Turkey
c
MedipolUniversity,DepartmentofOtorhinolaryngology,Istanbul,Turkey
dBezmialemVakıfUniversity,MedicalSchoolofHealthSciencesofAudiology,Istanbul,Turkey
1. Introduction
The mostfrequent reasonsforhearing lossare age,genetic factors,medicineototoxicityandacoustictrauma. Thecellular mechanism of hearing loss due to loud noise is not clearly understood.Constantexposuretohighintensityacoustictrauma results in death of the outer hair cells of the organ of Corti throughapoptosis[1].Thecausesofcelldeathduetoacoustic traumaarebloodflowreductionintheinnerear[2],freeradicals producedduetoincreasedmetabolicactivity[3,4],andcellular necrosisin theouterhaircellsdirectlyinducedbymechanical trauma[5].
Inlossofhearingduetoacoustictrauma,thereactiveoxygen radicalsplaytheroleofaprimerbycreatinganapoptoticsignalin theouterhairycells.Ithasbeenobservedthathydroxylradicals increaseupto10timesinthecochleaofratsthatareconstantly exposedtoacoustictrauma[3].Otherstudieshavealsoshownthat thenumber of reactive oxygen radicals in animals exposed to
acoustictraumaincreaseuptofourtimescomparedtoanimalsnot exposedtotrauma[6,7].
Medicalozonetherapyisusedforthetreatmentof inflamma-tion,infectedwounds,chronicskindiseaseandadvancedischemic illnesses,includingburns,duetoitsantioxidant,antiflammatory andantimicrobialeffects.Pureozoneisnotusedinozonetherapy owingtoozone toxicity;it is appliedin theformof anozone/ oxygen mixture[8,9].Ozonegas(O3)isproducedfromthesun
usingtheeffectsofultravioletrays,oritisproducedartificially usinganozonegenerator[10].
Medical ozone treatment is a method in which a gas combination containing ozone and oxygen is applied to body liquidsand cavities.Ithasbeenobservedthat ozonetreatment significantlydecreasesoxidativestressinexperimentalratmodels
[11,12].
It has been observed that oxidative stress is reduced with reinfusionofbloodmixedwithozonesinceitincreasesnitricoxide (NO) levels and results in a reduction in hypoxia due to vasodilatation in ischemic areas, superoxide dismutase (SOD) activationand a reduction inglutathione levels[13,14].During infusionof ozonizedblood totherecipient,themajority ofthe endothelialcellsareactivatedwithlipidoxidationproducts(LOPs), and this results in increased NO, plasma S-nitrosothiol and S-nitrosohemoglobinproduction.Althoughthehalf-lifeofNOisless
ARTICLE INFO
Articlehistory:
Received21January2013
Receivedinrevisedform21June2013 Accepted22June2013
Availableonline19July2013
Keywords: Acoustictrauma Ozonetherapy Innerear
Otoacousticemission Auditorybrainstemresponse
ABSTRACT
Objectives:Thegoalofthestudywastolookatthepotentialprotectiveeffectofozonetherapyby studyingitsantioxidantandvasodilatationeffectsagainsthearinglosscausedbyacoustictrauma. Methods:Thirty-twomaleWistarAlbinoratsweredividedintofourgroupsofeight.The1stgroupwas exposedtoacoustictrauma,the2ndgroupwastreatedwithozoneinitially,andwasexposedtoacoustic trauma24hlater,the3rdgroupreceivedozonewithouttrauma,whilethe4thgroupwasthecontrol group.The1stand2ndgroupswereexposedtoacoustictraumawith105dBSPLwhitebandnoisefor 4h.DPOAEandABRtestswereconductedinallgroupsonthe1st,5th,and10thdaysaftertrauma. Results:Inthe1stgroup,theeffectsofacoustictraumacontinuedondays1,5and10.The2ndgroup’s DPOAEandABRresultsondays5and10showedsignificantimprovementatallfrequenciescomparedto deteriorationonday1,andthereadingswerecomparabletobaselinemeasurements.
Conclusion:Acoustictraumaisapathologythatisexperiencedfrequentlyandleadstomanyproblemsin termsofhealthandcost.Ozonewasdemonstratedtobeareparativesubstanceagainstacoustictrauma and,inaddition,itcanbesuppliedandappliedeasily.
ß2013ElsevierIrelandLtd.Allrightsreserved.
*Correspondingauthorat:KaramanStateHospital,OtorhinolaryngologyClinic, TurgutO¨ zalStreetNo.1,Karaman,Turkey.Tel.:+905055040696;
fax:+903382263309.
E-mailaddress:alperyenigun@gmail.com(A.Yenigun).
ContentslistsavailableatSciVerseScienceDirect
International
Journal
of
Pediatric
Otorhinolaryngology
j ou rna l h ome pa ge : w ww . e l se v i e r. co m/ l oc a te / i j porl
0165-5876/$–seefrontmatterß2013ElsevierIrelandLtd.Allrightsreserved.
than1s,NOconnectedtoproteinmayinducevasodilatationinthe farischemicvascularfield[15].
Thisresearchhasbeenconductedtostudytheantioxidantand vasodilationeffectsofozonetherapy againstdamagecausedby acoustic trauma that results in reactive oxygen radicals and vasoconstrictionintheinnerear.
2. Materialsandmethods 2.1. Animals
Thestudywasconductedafterapproval(approvalno.2011/65) had been obtained from the Animal Experiments Local Ethics BoardofBezmialemVakifUniversity.Thirty-twohealthymature maleWistar Albinorats,weighing200–240g,wereusedin the study. All rats were evaluated otoscopically, and those with pathologicfindings(serousotitis,acuteotitis,adhesiveotitis,etc.) were excluded from the study. All rats were housed in an environmentwithatemperatureof2118C,witha12hlight,12h darkcycle,wheretheyhadfreeaccesstofoodandwater,andwhere thebackgroundnoiselevelwasbelow50dB(Table 1).Theratswere sacrificedonthe10thdayofthestudy.Theirmalondialdehyde(MDA;
m
mol/L),superoxidedismutase(SOD;U/ml)andadvancedoxidative proteinproduct(AOPP;m
mol/L)levelsweremeasuredusingblood samplesobtainedbeforesacrification.2.2. Hearingassessment
At the beginning of the study, the pinna reflex test was performedforhearingassessmentofallrats.Ketamine45mg/kg i.m. was used to induce sedation, after which all rats were examinedotoscopically.Anyobstacleswhichmight impedethe tests,suchasearwax,wereremoved.Then,thebasal Distortion-ProductOtoacoustic Emission(DPOAE) and Auditory Brainstem Response(ABR)measurementswereperformedonallrats. 2.2.1. DPOAE
A GSI Audera otoacoustic emission instrument was usedfor DPOAEmeasurements.Thesmallestsizetympanometryprobewas attachedtothetipofthedevice.Measurementswerecarriedoutina noise-treatedcabin.Themeasurementprocesswasinitiatedafter observingthattheearprobeofthedevicewasintheappropriate measurement position with proper configuration of its probe indicatorandstimulationwaveform.DPOAEsweremeasuredusing stimulations with different frequencies and intensities. Primary signallevels were adjustedtoL1=65dB,L2=55dBforDPgram measurements.Frequenciesoftheprimarysignalsweresetas1.2. DPgrammeasurementswerecarriedoutat2997,4002,5004,6002, 7001, 8003,9006, 10005, 11000and 12000Hz frequenciesas a functionoff2.Thedetectionthresholdwasdefinedastheprimary signallevelatwhichtheDPOAEwasjustdistinguishable,at3dB abovethe noise floor. In allmeasurements,the responseswere recordeduptothehighestlevelandthetestwasconcluded. 2.2.2. ABR
A Viasys Medelec Synergy instrument was used for ABR measurements.Measurements wereperformed on both earsof
theanesthetizedratsinanoise-treatedcabin.ABRresponseswere recorded using needle electrodes placed under the skin. The electrodes were positioned as follows: active electrode in the vertex,groundelectrodeinthecontralateralmastoidandreference electrode in the ipsilateralmastoid. The stimuliwere provided through insert earphones using 8kHz tone-burst sounds with alternatingpolarities.Thefilterwasadjustedto30–3000Hz,the repetitionratewas21/s and thetimewindow wasadjustedto 10ms.Atotalof1024stimuliweregivenforsignalaveraging.
Thethreshold wasdefined asthelowestintensitylevel that could beobserved and repeated. Each test started by applying stimuliat80dBnHLlevelandtheintensitywasreducedin20dB stepsuntilthethresholdvaluewaspassed.Asweapproachedthe threshold,we preferredtoincrease theintensityby10dBeach timeuntilwereachedthethresholdvalue.Atleasttwotraceswere createdforeachmeasurement,andattemptsweremadetorepeat thethresholdtocross-checkit.TheABRthresholdwasdefinedas thelowestintensitylevelinwhichwaveIIIofABRwasobserved. Baseline ABR measurementscarriedoutbefore acoustictrauma werecomparedwithABRmeasurementsonthe1st,5thand10th daysaftertrauma.
2.3. Noiseexposureandprocedures
Thefirsttwogroupsofratswereexposedtoacoustictrauma, using105dBSPL(soundpressurelevel)whitenoisefor4h.The ratsweresedatedwithanintramuscularinjectionofketamine 45mg/kg,andDPOAEandABRmeasurementswerecarriedout on the1st, 5thand 10thdays following acoustictrauma. The measurementswerecarriedoutinaroomwherethenoiselevel didnotexceed50dB.
2.4. Ozoneapplication
Ozone(O3)gasat0.7mg/kgwasappliedtoGroup2and3rats
throughintraperitonealinjection24hbeforethetraumaandwas continuedfor10daysaftertheacoustictrauma.Forthispurpose, the concentration of ozone produced by the ozone generator (Ozonosanphotonik,DrJ.Ha¨nslerGmbH,Iffezheim,Germany)was adjustedto75
m
g/ml.Theaveragevolumeoftheoxygen/ozonegas mixturecontainingO3gaswith0.7mg/kgdosagewasidentifiedtobe5mlforratsweighingbetween200and240g.Anequivalent volume (5ml) of salinewas intraperitoneally injected into the controlgrouprats.
2.5. Biochemicalparameters
Bloodsamplesextractedintracardiacallyfromtheratsonthe 10thdaywerecentrifugedat3500cycles/minandtheserumwas separated.Theserumsampleswerestoredat 808CinEppendorf tubeswithclosedcaps,andtaggedwithnumbersuntilbiochemical analyses were conducted. On the day of the analysis, serum sampleswereleftatroomtemperatureuntiltheyhadmelted,after whichMDA,SODandAOPPenzymeactivitiesweremeasured. 2.5.1. MeasuringMDALevel
ForMDAmeasurement,citratedbloodplasmawasseparated from the tube and stored at 808C. The MDA levels were determinedusingacolorspectrophotometerafterthiobarbituric acidhadreactedwithMDAatawavelengthof532nm.Thevalues obtainedthroughthismeasurementwereprovidedinnmol/ml. 2.5.2. Measuringsuperoxidedismutaseactivity(SOD)
Asuperoxidedismutasemeasurementkit(CaymanSuperoxide DismutaseAssayKit,CaymanChemicalCompany,USA)wasused for this purpose. The principle of the method is based on
Table1
Experimentalgroups.
Groups Procedure No.ofrats Group1 Acoustictrauma 8 Group2 Acoustictraumaandozonetherapy 8 Group3 Ozonetherapy 8 Group4 Controlgroup 8
identificationof the superoxide radicals produced by xanthine oxidaseandhypoxanthine,usingtetrazoliumsalt.1unitofSOD enzymeactivityisdefinedasthequantityofenzymerequiredfor dismutationof50%ofthesuperoxideradicals.Totalactivityofthe threetypesofSOD(Cu/Zn-,Mn-,Fe-SOD)wasmeasuredusingthis method.SODenzymeactivitywasprovidedasU/mL.
2.5.3. MeasuringserumAOPPlevel
AOPP formation is induced by the formation of chlorine oxidants(suchaschloraminesandhypochlorousacid).Thus,the AOPPconcentrationvariesinrelationtothosesubstances.Because ofthiscorrelation,Chloramine-Tinitsstandardformwasusedto measuretheAOPPconcentrations.Thetemperatureofthesamples was adjusted to room temperature before the study, and the sampleswerethenprocessedinaLinear CromalinePhotometry device.Forthispurpose,160
m
LofPBS(phosphate-bufferedsaline) wasadded to10m
L ofserumand mixed,and themixture was incubatedfor25s.20m
Lofaceticacidwasthenadded,andthe mixturewasincubatedfor25s.Finally,10m
LofKIsolutionwas added and the mixture was incubated for another 25s, and absorbance was read at 340nm. Serum which exceeded the linearity limit was diluted before processing. Measured AOPP concentrationswere determinedwithChloramine-Tin units ofm
mol/L.Allstepswerecarriedoutat378C,andtimeintervalswere adjustedto25sorlongerforeachstep.2.6. Sacrificeandenucleation
All experimental animals weresacrificed with 200mg/kg of intracardiac thiopental. The same method of sacrifice and enucleationwasusedforallanimals.
2.7. Statistics
StatisticalanalysiswascarriedoutusingtheStatisticalPackage fortheSocialSciencesversion13.0forWindows(SPSSInc,Chicago, IL,USA).Allquantitativevariableswereestimatedusingmeasures of central location (i.e. mean and median) and measures of
dispersion (i.e. standard deviation (SD)). Data normality was checkedusingtheKolmogorov–Smirnov testfor normality.The Student’s t-test was used to compare averages in normally distributed data within the four groups. The ANOVA test was usedtocomparetheABRthresholdvalues betweenthegroups. p<0.05wasacceptedasstatisticallysignificant.
3. Results 3.1. DPOAE
Group1(traumaonly):16earsofeightratswereevaluatedin Group1.SignificantdifferenceswereobservedbetweentheDPOAE valuesmeasuredbeforeandfollowingexposuretoacoustictrauma (p=0.003).Nosignificantdifferencewasidentifiedbetweenthe DPOAEvaluesmeasuredonthe1stdayafteracoustictrauma,and thosemeasured onthe5th and10thdays(p=0.564; p=0.644) (Fig. 1).
Group2(trauma+ozone):16earsofeightratswereevaluatedin Group2.SignificantdifferenceswereobservedbetweentheDPOAE valuesmeasuredbeforeandfollowingexposuretoacoustictrauma (p=0.012).Significantdifferenceswerealsoobservedbetweenthe DPOAEvaluesmeasuredonthe5thand 10thdaysafterexposure totrauma(p=0.02;p=0.03)(Fig. 1).
Group3(ozoneonly):16earsofeightratswereevaluatedin Group 3. No significant difference was observed between the DPOAE values measured ozone treatment (p=0.140; p=0.396; p=0.489)(Fig. 1).
Group4(controlgroup):16earsofeightratswereevaluatedin Group 4. No significant difference was observed between the baselinemeasurementsandtheDPOAEvaluesmeasured onthe 1st,5thand10thdays(p=0.791;p=0.965;p=0.945)(Fig.1). 3.2. ABR
Group 1 (trauma only): The ABRthreshold value increased significantly after acoustic trauma exposure (p=0.002). No significantdifferencewasidentifiedbetweentheABRthreshold
values measured on the 1st day after acoustic trauma, and thosemeasuredonthe5thand10thdays(p=0.526;p=0.672) (Table 2).
Group2(trauma+ozone):TheABRthresholdvalueincreased significantly after acoustic trauma exposure (p=0.003). A significant difference was also observed between the ABR thresholdvaluesmeasured beforetraumaandthosemeasured onthe5thand10th daysafter exposuretotrauma (p=0.003; p=0.003)(Table 2).
Group3(ozoneonly):Nosignificantdifference wasobserved between the ABR threshold values measured before ozone treatmentandthosemeasuredonthe1st,5thand10thdaysof treatment(p=0.462;p=0.965;p=0.532)(Table 2).
Group 4 (control group): No significant difference was observed between the baseline measurements and the ABR thresholdvaluesmeasuredonthe1st,5thand10thdaysinthe controlgrouptreatedwithsaline(p=0.972;p=0.947;p=0.432) (Table2).
3.3. ComparisonofABRthresholdsamongthegroups
No significant difference was observed between the ABR thresholdsofthe1stand2ndgroupsonthe1stdayafteracoustic trauma(p=0.423)(Fig. 2).Asignificantdifferencewasdetected betweentheABRthresholdsofthe1stand2ndgroupsmeasured on the 5th and 10th days after exposure to acoustic trauma (p=0.003;p=0.003)(Fig. 2).
AsignificantdifferencewaspresentbetweenGroups1and3 alsoGroups1and4fortheABRthresholdvaluesonthe1st,5th and 10th days (p=0.002) (Fig. 2). The difference was not significantbetweenGroups3and4onthe1st,5thand10thdays (p=0.950;p=0.593;p=0.946)(Fig. 2).
AsignificantdifferencewaspresentbetweenGroups2and3 alsoGroups2and4fortheABRthresholdvaluesonthe1stday (p=0.004;p=0.004)(Fig.2).Thedifferencewasnotsignificant between Groups 2 and 3 also Groups 2 and 4 for the ABR thresholdvaluesonthe5thand10thdays(p=0.850;p=0.850) (Fig.2).
3.4. Biochemicalparameters
TheMDAvaluesofthebloodtestsofGroup2(trauma+ozone) weresignificantlylessthanthoseofGroup1(traumaonly),andthe SOD values of Group2 were significantlyhigher (p<0.05). No significantdifferencewasobservedbetweenGroup3(ozoneonly) andGroup4(controlgroup)forMDAvalues(p=0.512),andSOD values(p=0.112).Nosignificantdifferencewasobservedbetween Group1 (trauma only)andGroup2 (trauma+ozone)for AOPP values(p=0.06)(Fig. 3).
4. Discussion
Thepathogenesisofacoustictraumaismultifactorial.Acoustic traumamayresultindamagetohaircells,disordersofstereocilia, collapseofsupportcells,ruptureofdendritesandstructuraland metabolic damage such as strial edema [16,17]. Bray and associates have studied23diskjockeys. The noiselevel wasat least108dBintheir studyenvironmentand theyobservedthat 70%complainedoflossofhearingand74%complainedoftinnitus
[18].Thisstudywasconductedtoshowthepotentialprotective effect of ozone by examining the effects of antioxidants and vasodilatationagainsthearinglossduetoacoustictrauma.
Broadbandnoise(whitenoise),withaconstantamplitudeover the whole frequency spectrum, causes homogeneous damage throughout the cochlea [19]. In our study, we applied 105dB broadbandnoisetothefirsttwogroupsofratsfor4htocreate acoustictrauma.ABRandDPOAEmeasurementswerecarriedout onthe1stdayaftertraumainthe1stand2ndgroups;thesevalues, when compared withthebaseline values, showeda significant difference (ABR: Group1 p=0.002, Group 2 p=0.003;DPOAE: Group 1 p=0.003, Group 2 p=0.012), which confirmed that acoustictraumawassuccessfulininducinghearingloss.
NosignificantdifferencewasobservedbetweenGroup1and Group 2 on the1st day afteracoustic trauma (p>0.5), which shows thatinitially,ozonetreatment didnothavea preventive effectonacoustictrauma.However,a significantdifferencewas observed betweentheABR thresholdvalues of Groups1 and 2 measured on the 5th and 10thdays afterexposure toacoustic
Table2
Thresholdsofauditorybrainstemresponsemeasuredbeforeacoustictraumaand1,5,and10daysaftertrauma.
Groups Before 1stday 5thday 10thday
Group1(trauma) 26.8 43.6(p=0.002)¥
42.5(p=0.526)C 40.2(p=0.672)C Group2(trauma+ozone) 28.9 44.3(p=0.003)¥ 32.5(p=0.003)¥ 31.3(p=0.003)¥
Group3(ozone) 27.1 27.5(p=0.462)C 28.7(p=0.965)C 26.2(p=0.532)C Group4(control) 29.1 31.3(p=0.972)C 29.3(p=0.947)C 29.4(p=0.432)C Student’st-testforindependentsamples(p<0.05).
¥
Significancelevelobtained.
C
Insignificantlevelobtained.
Fig.3.Biochemicalparameters. Fig.2. Comparisonof ABR thresholds among the groups.While there is no
statisticallysignificantdifferencebetweenGroups1and2aftertrauma(p>0.01), thereisasignificantdifferenceonthe5thand10thdaysforABRthresholdvalues (p<0.01).
trauma (p=0.003; p=0.003). In Group 2, with ozone given prophylactically, the ABR and DPOAE values on the 5th and 10thdayswererestoredtothebaselinevalues,andtheeffectsof theestablished acoustictrauma werecompletelyremoved and corrected.Thesefindingsshowthat,afteracoustictrauma,ozone repairsthedamageintheinnerear.Inourstudy,no significant differencewasidentifiedbetweenGroups3and4onthe1st,5th and10thdays(p=0.950;p=0.593;p=0.946).Thisdemonstrates thatozonehasnonegativeeffectonhearing.Thisisthefirststudy inthemedicalliteraturetoinvestigatetherelationshipbetween ozonegivenprophylacticallyanditsototoxicity,andwhichshows therapidreparativeeffectofozonetodamagecausedbyacoustic trauma.
We measuredserumMDA,SODand AOPPlevelstoexamine oxidativestressrelatedtoacoustictrauma.MDAvaluesinGroup2 (trauma+ozone)weresignificantlylowerthaninGroup1(trauma only)(p<0.05), and SOD values in Group2 were significantly higher than in Group 1 (p<0.05). No statistically significant differencewasobservedbetweentheAOPPvaluesofGroups1and 2.Ourstudyisagainthefirstinthemedicalliteraturetoinvestigate therelationshipbetweenAOPPandacoustictrauma.Thefinding that, while oxidative parameters increased after exposure to acoustictrauma,nopermanenthearinglosswasseeninthelong term,makingit necessarytoconductfurther researchonother factors(antioxidantenzymesandheatshockproteins)whichmay leadtohearing lossorwhichmaylimit theeffects ofoxidative stress.
Thetreatmenteffectofozoneis,inparticular,duetoreactive oxygen products (ROP),hydrogen peroxide and Lipid oxidation products(LOP).Whenozonizedbloodisre-infusedtotheblood circulation,itactivatesendothelialcellsbyLOP,andnitricoxide, plasmaS-nitrosothiolandS-nitrosohemoglobinareproduced.The half-lifeofnitricoxideisless than1s,butthehalf-lifeofnitric oxideconnectedtoproteinis muchlonger, andthis maycause vasodilatationindistantregions[9].Ithasbeenproventhatlow doseozoneisusefulininducingantioxidantenzymes,nitricoxide canalandothercellularactivities[20].
Ozone therapy applied to an ischemic organ results in the creation of erythrocytes, increased oxygen absorption in these erythrocytesandemissionofnitricoxide,carbonmonoxideand growthfactorsfrom endothelialcells and thrombocytes, which increasesoxygenationfurther[9].Althoughtheamountofoxygen that enters the body with infusion of blood ozonizedwith an oxygen/ozone mixture may be negligible, ozone changes the normaloxygendistributionlevelbytriggeringaseriesofbiologic events,anditshowsitseffectsinthisway.Asaresult,ozonehas beenused for treatment, since it hasbeen thoughtthat it can preventischemia-reperfusiondamage[21,22].
Ozone treatment leads to free radical production such as hydrogen peroxide (H2O2), reactive oxygen radicals (ROS), and
lipid oxidation products (LOP). Antioxidation enzymes that provide system defense (superoxide dismutase (SOD), catalase (CAT),andglutathioneperoxidase(GSH-Px))arestimulatedtoward
these increased free radicals. In studies using ozone, this stimulationisreferredtoasoxidativepreconditioning.Thescope ofthesestudiesistocreateischemia-likeconditionssothatthe bodydefensesystemisstimulated[9,20].Ithasbeenshownthat controlled ozone therapy can decreasethe damageinduced by reactiveoxygenproductionbyprovidingadaptationtooxidative stress [23]. Ozone treatment with preconditioning by creating ischemia-likeconditionsisa viablemethodin thetreatment of ischemic vascular diseases by stimulating the synthesis of antioxidationenzymes[24].In ourstudy,antioxidationenzyme synthesiswasincreasedwithstressadaptationbystartingozone treatment one day before acoustic trauma and generating preconditioningusingozonefreeradicals.
In recent studies, it was shown that ozone preconditioning treatment is an effective way to prevent ischemia perfusion damagein variousorganssuchastheliver,lungs,andkidneys. Ajamieh et al. [21] have demonstrated by histopathologic examinationthatozonepreconditioningaidsrecoveryinhepatic ischemia.Inanotherstudy,itwasshownthatcreatingoxidative preconditioningwithozonecanstimulatetheendogen antioxida-tionsystemagainstliverdamagebycreatingoxidativestress[24]. Barberetal.[22]havereportedthatozonepreconditioningbefore renaltransplantationdecreasedrenaldamageinratswithrenal ischemia.Stadlbaueret al.haveappliedintraperitonealozoneto both donor and receiver before cardiac transplantation and demonstratedbyhistologicand biochemicaloutcomesthatthis ozone application led to a reduction in ischemia reperfusion damageoccurringduringtransplantation[25].Moreover,Borrego etal.[26]haveshownthatozonehasaneffectontheantioxidation systeminratsandreducesthedamagecausedbyinducedrenal ischemiawithcisplatin.Accompanyingthesestudies,inourstudy, wehavenotcomeacrossanypathologicfindingintheliver,lung, and kidney during autopsy after sacrifice and because of this, additionalexaminationwasnotrequired.However,furtherstudies shouldbeperformedtoclarifyanypossiblesideeffectsofozoneon othersystemsandorgans.
Ithasbeensuggestedthatozonetherapymaybeabletoprotect antioxidantsystemsandkeepotherindicatorsofendothelialcell damage,whicharerelatedtodiabeticcomplications,atphysiologic levels since there is a known relationship between diabetes mellitusandoxidativestress[27].
H1-receptor antagonists, corticosteroids, vasodilators, antic-oagulants,volumeexpanders,antioxidantagentssuchasvitamin A,Cand E,and many otheragents suchasMg and hyperbaric oxygen,havebeenemployedinthetreatmentofacoustictrauma, eitherisolatedorinvariouscombinations[28].Inourstudy,we believethatOzone’sbenefitswereobservedinasshortatimeas5 days,duetoitsbiphasiceffectwhichactivatesvasodilatoragents andtheendogenousantioxidantsystem.
In papers showing the relationship between ketamine and neuronal toxicity, it was demonstrated that ketamine causes neuronalcelldeathifitisadministeredassixorseveninjectionsof 20mg/kg.Alowernumberinjectionsdidnotcauseanyneuronal damage[29,30].Inaddition,anotherstudyshowedthatasingle doseofketamine(25,50,or75mg/kg)didnotcauseanyneuronal degeneration.However,repeateddosesof25mg/kgketamineat 90min intervals over 9h increased neuron degeneration [31]. Moreover,ketaminedoseforratsisstatedas40–80mg/kgthrough IM at rodent anesthesia and analgesia guideline prepared by UniversityofPennsylvania[32].
For this reason, we have used a single dose of 45mg/kg ketamine in our study, and have not observed any neuronal pathologyaffectingtheresults.
5. Conclusion
Acoustictraumaiscurrentlyafrequentlyencountered pathol-ogy,whichleadstoeconomiccostsandhealthproblems.Ozone given pre-exposure may prevent permanent hearing loss from acoustictrauma,duetoitsbiphasiceffect,whilebyitself,ozone hasantioxidantand vasodilatoryproperties.Thisstudy demon-stratedthatozonemayberegardedasanalternativetherapythat isconvenientlyaccessible,andisaneasilyapplicableotoprotective substancerepairinginnereardamageinducedbyacoustictrauma inrats.
Funding None.
Conflictofinterest None.
Acknowledgements
TheauthorswouldliketothankAss.Prof. OmerUysal,PhD, fromtheDepartmentofBiostatisticsandMedicalInformaticsfor hiskindstatisticalassistance.
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